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"schema_version": "seqcolyte.spec.v1",
"spec_id": "drop_seq",
"assay": "Drop-seq",
"chemistry_version": "unspecified",
"platform": "illumina",
"platform_params": {
"read_type": "short"
},
"source_docs": [
{
"doc_id": "Drop-seq_author_protocol_v1.1.pdf",
"title": "Drop-seq Laboratory Protocol v1.1",
"url": "https://doi.org/10.17504/protocols.io.mkbc4sn",
"path": "/Users/seqmachines/playground/protocols-test/protocols/drop_seq/Drop-seq_author_protocol_v1.1.pdf",
"retrieved_date": null
},
{
"doc_id": "drop-seq_supp.pdf",
"title": "drop-seq_supp.pdf",
"url": null,
"path": "/Users/seqmachines/playground/protocols-test/protocols/drop_seq/drop-seq_supp.pdf",
"retrieved_date": null
},
{
"doc_id": "drop-seq_paper.pdf",
"title": "drop-seq_paper.pdf",
"url": null,
"path": "/Users/seqmachines/playground/protocols-test/protocols/drop_seq/drop-seq_paper.pdf",
"retrieved_date": null
}
],
"oligos": [
{
"oligo_id": "oligo_barcoded_bead_seqb",
"name": "Barcoded Bead SeqB (bead capture oligo)",
"aliases": [],
"role": "Bead-tethered capture/RT primer: SMART PCR handle + cell barcode + UMI + oligo-dT (recommended go-forward bead; used in retina replicates 4-7)",
"kind": "assembled",
"sequence": "TTTTTTTAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
"direction": "5_to_3",
"components": [
{
"name": "Bead-Linker",
"sequence": "",
"role": "18-carbon PEG flexible-chain linker tethering the oligo to the Toyopearl bead (sequence not specified)"
},
{
"name": "T-spacer",
"sequence": "TTTTTTT",
"role": "spacer"
},
{
"name": "SMART PCR handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
"role": "constant PCR/sequencing handle (SeqB variant, ends ...GAGTAC)"
},
{
"name": "Cell barcode",
"sequence": "[CELL_BARCODE:12]",
"role": "12-bp split-and-pool cell barcode (JJJJJJJJJJJJ; 4^12 = 16,777,216 sequences)"
},
{
"name": "UMI",
"sequence": "[UMI:8]",
"role": "8-bp unique molecular identifier (NNNNNNNN; 4^8 = 65,536)"
},
{
"name": "Poly(dT)",
"sequence": "TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
"role": "30-nt oligo-dT mRNA capture / RT priming"
}
],
"provenance": "reagent",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6. 5'-Bead-Linker-TTTTTTTAAGCAGTGGTATCAACGCAGAGTAC JJJJJJJJJJJJ NNNNNNNN T(30)-3'. Prime Read 1 with Read1CustomSeqB. >10^8 primers per bead."
},
{
"oligo_id": "oligo_barcoded_bead_seqa",
"name": "Barcoded Bead SeqA (bead capture oligo)",
"aliases": [],
"role": "Bead-tethered capture/RT primer variant with a longer handle (...GAGTACGT); used in the human-mouse experiments and retina replicates 1-3",
"kind": "assembled",
"sequence": "TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
"direction": "5_to_3",
"components": [
{
"name": "Bead-Linker",
"sequence": "",
"role": "18-carbon PEG flexible-chain linker (sequence not specified)"
},
{
"name": "T-spacer",
"sequence": "TTTTTTT",
"role": "spacer"
},
{
"name": "SMART PCR handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGTACGT",
"role": "constant PCR/sequencing handle (SeqA variant, ends ...GAGTACGT)"
},
{
"name": "Cell barcode",
"sequence": "[CELL_BARCODE:12]",
"role": "12-bp split-and-pool cell barcode (JJJJJJJJJJJJ)"
},
{
"name": "UMI",
"sequence": "[UMI:8]",
"role": "8-bp UMI (NNNNNNNN)"
},
{
"name": "Poly(dT)",
"sequence": "TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT",
"role": "30-nt oligo-dT"
}
],
"provenance": "reagent",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6. 5'-Bead-Linker-TTTTTTTAAGCAGTGGTATCAACGCAGAGTACGT JJJJJJJJJJJJ NNNNNNNN T(30)-3'. Prime Read 1 with Read1CustomSeqA."
},
{
"oligo_id": "oligo_template_switch_oligo",
"name": "Template_Switch_Oligo (TSO)",
"aliases": [],
"role": "Template-switching oligo; its 3\u2032 riboG triplet pairs the untemplated C's added by Maxima H- RT, appending the SMART handle to the cDNA 3\u2032 end",
"kind": "single",
"sequence": "AAGCAGTGGTATCAACGCAGAGTGAATGGG",
"direction": "5_to_3",
"components": [
{
"name": "SMART handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGT",
"role": "constant handle"
},
{
"name": "Spacer",
"sequence": "GAAT",
"role": "spacer"
},
{
"name": "Riboguanosines",
"sequence": "GGG",
"role": "3\u2032 rGrGrG that base-pairs with cDNA 3\u2032 dC overhang"
}
],
"provenance": "reagent",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6: AAGCAGTGGTATCAACGCAGAGTGAAT rGrGrG (folded rGrGrG -> GGG). Used at 2.5 \u00b5M (supp) / 50 \u00b5M stock (protocol) in RT."
},
{
"oligo_id": "oligo_tso_pcr_smart_primer",
"name": "TSO_PCR / SMART PCR Primer (Template_Switch_PCR primer)",
"aliases": [],
"role": "Whole-transcriptome cDNA amplification primer; anneals to the SMART handle present on both cDNA ends after template switching",
"kind": "single",
"sequence": "AAGCAGTGGTATCAACGCAGAGT",
"direction": "5_to_3",
"components": [
{
"name": "SMART handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGT",
"role": "PCR primer / handle"
}
],
"provenance": "reagent",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6 TSO_PCR. Called 'SMART PCR PRIMER' (100 \u00b5M) in the protocol; 0.8 \u00b5M in supp."
},
{
"oligo_id": "oligo_p5_tso_hybrid",
"name": "P5-TSO_Hybrid (New-P5-SMART PCR hybrid oligo)",
"aliases": [],
"role": "Library-PCR forward primer after tagmentation; appends the Illumina P5 adapter to the SMART/3\u2032-cDNA end",
"kind": "assembled",
"sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC",
"direction": "5_to_3",
"components": [
{
"name": "P5",
"sequence": "AATGATACGGCGACCACCGAGATCTACAC",
"role": "Illumina P5 adapter"
},
{
"name": "Bridge",
"sequence": "GCCTGTCCGCGG",
"role": "linker/bridge sequence"
},
{
"name": "SMART handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
"role": "anneals to bead SMART handle; ends with phosphorothioate *A*C"
}
],
"provenance": "reagent",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6 P5-TSO_Hybrid: AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT*A*C (3\u2032 *A*C = phosphorothioate). Used at 200 nM in Nextera PCR."
},
{
"oligo_id": "oligo_nextera_n701",
"name": "Nextera_N701 (N70X i7 library primer)",
"aliases": [],
"role": "Library-PCR reverse primer after tagmentation; appends the i7 sample index + Illumina P7 adapter to the Nextera (Tn5) end",
"kind": "assembled",
"sequence": "CAAGCAGAAGACGGCATACGAGATTCGCCTTAGTCTCGTGGGCTCGG",
"direction": "5_to_3",
"components": [
{
"name": "P7",
"sequence": "CAAGCAGAAGACGGCATACGAGAT",
"role": "Illumina P7 adapter"
},
{
"name": "i7 sample index (N701)",
"sequence": "TCGCCTTA",
"role": "8-bp i7 sample index"
},
{
"name": "Nextera Read 2 handle (s7)",
"sequence": "GTCTCGTGGGCTCGG",
"role": "anneals to Nextera mosaic-end/s7 region installed by tagmentation"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6. Condition-1 index in species-contamination experiment. Used at 200 nM in Nextera PCR."
},
{
"oligo_id": "oligo_nextera_n702",
"name": "Nextera_N702 (i7 library primer)",
"aliases": [],
"role": "Alternate i7-indexed library-PCR reverse primer (species-contamination condition 2)",
"kind": "assembled",
"sequence": "CAAGCAGAAGACGGCATACGAGATCTAGTACGGTCTCGTGGGCTCGG",
"direction": "5_to_3",
"components": [
{
"name": "P7",
"sequence": "CAAGCAGAAGACGGCATACGAGAT",
"role": "Illumina P7 adapter"
},
{
"name": "i7 sample index (N702)",
"sequence": "CTAGTACG",
"role": "8-bp i7 sample index"
},
{
"name": "Nextera Read 2 handle (s7)",
"sequence": "GTCTCGTGGGCTCGG",
"role": "s7 region"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6."
},
{
"oligo_id": "oligo_nextera_n703",
"name": "Nextera_N703 (i7 library primer)",
"aliases": [],
"role": "Alternate i7-indexed library-PCR reverse primer (species-contamination condition 3)",
"kind": "assembled",
"sequence": "CAAGCAGAAGACGGCATACGAGATTTCTGCCTGTCTCGTGGGCTCGG",
"direction": "5_to_3",
"components": [
{
"name": "P7",
"sequence": "CAAGCAGAAGACGGCATACGAGAT",
"role": "Illumina P7 adapter"
},
{
"name": "i7 sample index (N703)",
"sequence": "TTCTGCCT",
"role": "8-bp i7 sample index"
},
{
"name": "Nextera Read 2 handle (s7)",
"sequence": "GTCTCGTGGGCTCGG",
"role": "s7 region"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6."
},
{
"oligo_id": "oligo_read1_custom_seq_a",
"name": "Read1CustomSeqA (custom Read 1 sequencing primer)",
"aliases": [],
"role": "Custom Read 1 sequencing primer for libraries made with Barcoded Bead SeqA; primes the cell barcode + UMI read",
"kind": "single",
"sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT",
"direction": "5_to_3",
"components": [
{
"name": "Read1CustomSeqA",
"sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACGT",
"role": "custom Read 1 primer"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6. Loaded at 0.3 \u00b5M for Read 1 priming on NextSeq/MiSeq."
},
{
"oligo_id": "oligo_read1_custom_seq_b",
"name": "Read1CustomSeqB (custom Read 1 sequencing primer)",
"aliases": [],
"role": "Custom Read 1 sequencing primer for libraries made with Barcoded Bead SeqB; primes the cell barcode + UMI read",
"kind": "single",
"sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC",
"direction": "5_to_3",
"components": [
{
"name": "Read1CustomSeqB",
"sequence": "GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC",
"role": "custom Read 1 primer"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6."
},
{
"oligo_id": "oligo_p7_tso_hybrid",
"name": "P7-TSO_Hybrid",
"aliases": [],
"role": "P7-side hybrid PCR primer used in the bead-primer RT validation (soluble-RNA) experiment to amplify off the SMART handle",
"kind": "assembled",
"sequence": "CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC",
"direction": "5_to_3",
"components": [
{
"name": "P7",
"sequence": "CAAGCAGAAGACGGCATACGAGAT",
"role": "Illumina P7 adapter"
},
{
"name": "Index/bridge",
"sequence": "CGTGATCGGTCTCGGCGG",
"role": "index + bridge region"
},
{
"name": "SMART handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
"role": "anneals to SMART handle; 3\u2032 *A*C phosphorothioate"
}
],
"provenance": "reagent",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6: CAAGCAGAAGACGGCATACGAGATCGTGATCGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGT*A*C. Used at 400 nM with TruSeq_F."
},
{
"oligo_id": "oligo_truseq_f",
"name": "TruSeq_F (standard Illumina TruSeq primer)",
"aliases": [],
"role": "P5/TruSeq Read 1 primer used in the soluble-RNA validation PCR and to prime Read 1 for the synRNA molecular-barcode read",
"kind": "single",
"sequence": "AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT",
"direction": "5_to_3",
"components": [
{
"name": "P5",
"sequence": "AATGATACGGCGACCACCGAGATCTACAC",
"role": "Illumina P5 adapter"
},
{
"name": "TruSeq Read 1",
"sequence": "TCTTTCCCTACACGACGCTCTTCCGATCT",
"role": "TruSeq Read 1 sequence"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6: ...CCGATC*T (3\u2032 phosphorothioate T)."
},
{
"oligo_id": "oligo_cust_syn_rna_seq",
"name": "CustSynRNASeq",
"aliases": [],
"role": "Custom Read 2 sequencing primer used in the synRNA bead-barcode-homogeneity experiment to read the 12-bp cell barcode + 8-bp UMI",
"kind": "single",
"sequence": "CGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC",
"direction": "5_to_3",
"components": [
{
"name": "CustSynRNASeq",
"sequence": "CGGTCTCGGCGGAAGCAGTGGTATCAACGCAGAGTAC",
"role": "custom sequencing primer"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6."
},
{
"oligo_id": "oligo_umi_smart_dt",
"name": "UMI_SMARTdT",
"aliases": [],
"role": "In-solution RT primer (SMART handle + 9-nt UMI + oligo-dT) for the standard in-solution template-switch amplification comparison",
"kind": "assembled",
"sequence": "AAGCAGTGGTATCAACGCAGAGTAC[UMI:9]TTTTTTTTTTTTTTTTTTTTTTTT",
"direction": "5_to_3",
"components": [
{
"name": "SMART handle",
"sequence": "AAGCAGTGGTATCAACGCAGAGTAC",
"role": "constant handle"
},
{
"name": "UMI",
"sequence": "[UMI:9]",
"role": "9-nt UMI (NNNNNNNNN)"
},
{
"name": "Poly(dT)",
"sequence": "TTTTTTTTTTTTTTTTTTTTTTTT",
"role": "24-nt oligo-dT RT priming"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6: AAGCAGTGGTATCAACGCAGAGTAC NNNNNNNNN T(24)."
},
{
"oligo_id": "oligo_synrna",
"name": "synRNA (polyadenylated synthetic RNA spike-in)",
"aliases": [],
"role": "Polyadenylated synthetic RNA carrying a 20-nt molecular barcode; used to quantify bead primer sites and validate barcode readout",
"kind": "assembled",
"sequence": "CCUACACGACGCUCUUCCGAUCU[UMI:20]BAAAAAAAAAAAAAAAAAAAAAAAA",
"direction": "5_to_3",
"components": [
{
"name": "TruSeq-derived RNA handle",
"sequence": "CCUACACGACGCUCUUCCGAUCU",
"role": "RNA sequence homologous to TruSeq Read 1 region (ribo)"
},
{
"name": "Molecular barcode",
"sequence": "[UMI:20]",
"role": "20-nt degenerate molecular barcode (rN x20)"
},
{
"name": "Anchor + poly(A)",
"sequence": "BAAAAAAAAAAAAAAAAAAAAAAAA",
"role": "rB anchor + poly(A) tail (24 A)"
}
],
"provenance": "document",
"derivation": null,
"sequence_source": "llm_extracted_from_docs",
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "oligo / final library",
"method": "claude_llm_extraction"
}
],
"notes": "Table S6, given as ribonucleotides (folded rX -> X; RNA U retained). Read 1 (TruSeq primer) reads the 20-bp molecular barcode."
}
],
"final_library": {
"source_label": "Drop-seq final Illumina library (Barcoded Bead SeqB chemistry; P5-TSO_Hybrid + Nextera N701)",
"annotated_library_sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG",
"library_sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG",
"strands": [
{
"direction": "5_to_3",
"source_html": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG",
"source_sequence": "AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG"
},
{
"direction": "3_to_5",
"source_html": "TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATG[CELL_BARCODE:12]'[UMI:8]'AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA[CDNA]'GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[SAMPLE_INDEX:8]'TAGAGCATACGGCAGAAGACGAAC",
"source_sequence": "TTACTATGCCGCTGGTGGCTCTAGATGTGCGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATG[CELL_BARCODE:12]'[UMI:8]'AAAAAAAAAAAAAAAAAAAAAAAAAAAAAA[CDNA]'GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG[SAMPLE_INDEX:8]'TAGAGCATACGGCAGAAGACGAAC"
}
],
"annotation_lines": [
"AATGATACGGCGACCACCGAGATCTACAC = P5",
"GCCTGTCCGCGG = SMART/P5 hybrid bridge",
"AAGCAGTGGTATCAACGCAGAGTAC = SMART PCR handle (Read 1 primer region)",
"[CELL_BARCODE:12] = 12-bp cell barcode",
"[UMI:8] = 8-bp UMI",
"TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT = poly(dT) (30 nt)",
"[CDNA] = cDNA insert",
"CTGTCTCTTATACACATCT = Nextera Read 2 mosaic end (revcomp of ME)",
"CCGAGCCCACGAGAC = Nextera Read 2 handle (s7)",
"[SAMPLE_INDEX:8] = i7 sample index",
"ATCTCGTATGCCGTCTTCTGCTTG = reverse complement of P7"
],
"evidence": [
{
"source_doc": "protocol_docs",
"locator": "Drop-seq final Illumina library (Barcoded Bead SeqB chemistry; P5-TSO_Hybrid + Nextera N701)",
"method": "claude_llm_extraction"
}
]
},
"read_structure": {
"reads": [
{
"read": "R1",
"primer": "Read1CustomSeqB / Read1CustomSeqA (custom)",
"template": "bottom",
"cycles": 20,
"segments": [
{
"name": "Cell barcode",
"type": "barcode",
"order": 0,
"scored": true,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null,
"length": 12
},
{
"name": "UMI",
"type": "umi",
"order": 1,
"scored": true,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null,
"length": 8
}
]
},
{
"read": "I1",
"primer": "Illumina i7 index primer",
"template": "top",
"cycles": 8,
"segments": [
{
"name": "i7 sample index",
"type": "index",
"order": 0,
"scored": false,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null,
"length": 8
}
]
},
{
"read": "R2",
"primer": "Nextera Read 2 primer",
"template": "top",
"cycles": 50,
"segments": [
{
"name": "cDNA insert",
"type": "insert",
"order": 0,
"scored": true,
"provenance": null,
"whitelist_ref": null,
"constant_ref": null,
"notes": null
}
]
}
]
},
"library_generation": [
{
"step": 1,
"title": "mRNA capture on barcoded bead (droplet co-encapsulation + lysis)",
"summary": "A lysed cell's polyadenylated mRNA hybridizes by its poly(A) tail to the 30-nt oligo-dT of a co-encapsulated barcoded bead.",
"note": "The co-flow device pairs one cell with one bead in a ~1 nL droplet; lysis buffer (Ficoll/Sarkosyl/Tris/EDTA/DTT) releases mRNA that anneals to the bead-tethered oligo-dT.",
"product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT----->\n 3'- AAAAA......[mRNA] -5'"
},
{
"step": 2,
"title": "Reverse transcription with template switching (STAMP formation)",
"summary": "Maxima H- RT extends the bead primer into first-strand cDNA and adds untemplated dC's at the 3\u2032 end.",
"note": "Reverse transcription copies the mRNA into cDNA covalently attached to the bead (a STAMP); the terminal-transferase activity of Maxima H- appends 2-3 dC's to the cDNA 3\u2032 end.",
"product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCC----->\n 3'- ...........[mRNA template] -5'"
},
{
"step": 3,
"title": "Template switch appends SMART handle to cDNA 3\u2032 end",
"summary": "The TSO rGrGrG anneals to the cDNA dC overhang and is copied, placing a SMART handle on the cDNA 3\u2032 end.",
"note": "Both ends of the first-strand cDNA now carry the AAGCAGTGGTATCAACGCAGAGT SMART handle, enabling single-primer whole-transcriptome PCR.",
"product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCCATTCACTCTGCGTTGATACCACTGCTT -3'\n GGGTAAG......TGACGAA -5' (from TSO)"
},
{
"step": 4,
"title": "Exonuclease I treatment",
"summary": "Exonuclease I chews back bead primers that never captured an mRNA, leaving only extended STAMPs.",
"note": "Single-stranded, unextended oligos are degraded so they cannot participate in PCR; barcode-bearing extended primers (double-stranded at the 5\u2032 handle after RT) are protected.",
"product": "|--5'- ...AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCCATTCACTCTGCGTTGATACCACTGCTT -3'"
},
{
"step": 5,
"title": "Whole-transcriptome PCR (SMART/TSO_PCR primer)",
"summary": "A single SMART PCR primer amplifies full-length cDNA off both handle-bearing ends.",
"note": "Kapa HiFi + TSO_PCR/SMART primer (AAGCAGTGGTATCAACGCAGAGT) produce double-stranded, amplified cDNA averaging 1300-2000 bp.",
"product": "5'- AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CCCATTCACTCTGCGTTGATACCACTGCTT -3'\n3'- TTCGTCACCATAGTTGCGTCTCATG[CB']........[UMI'](A)30[CDNA']GGGTAAGTGAGACGCAACTATGGTGACGAA -5'"
},
{
"step": 6,
"title": "Nextera XT tagmentation (Tn5)",
"summary": "Tn5 fragments the amplified cDNA and inserts the Nextera mosaic-end adapter.",
"note": "Tagmentation adds the Nextera Read 2 / s7 handle to internal 3\u2032-biased fragments; only fragments still bearing the 5\u2032 SMART handle can later receive P5, restricting the library to 3\u2032 ends.",
"product": "5'- AAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8](T)30[CDNA]CTGTCTCTTATACACATCT -3'\n <-- Nextera mosaic end (ME) -->"
},
{
"step": 7,
"title": "Nextera library PCR (P5-TSO_Hybrid + Nextera N70X)",
"summary": "P5-TSO_Hybrid adds P5 at the SMART end and Nextera N70X adds the i7 index + P7 at the tagmented end.",
"note": "Because P5 is added only via the SMART handle, exclusively 3\u2032-end cDNA fragments are amplified into the final sequenceable library (~450-680 bp).",
"product": "5'- AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC[CELL_BARCODE:12][UMI:8]TTTTTTTTTTTTTTTTTTTTTTTTTTTTTT[CDNA]CTGTCTCTTATACACATCTCCGAGCCCACGAGAC[SAMPLE_INDEX:8]ATCTCGTATGCCGTCTTCTGCTTG -3'"
}
],
"library_sequencing": [
{
"read": "Read 1 (cell barcode + UMI)",
"primer": "Read1CustomSeqB (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) \u2014 Read1CustomSeqA for SeqA beads",
"template": "bottom",
"cycles": 20,
"note": "Custom Read 1 primer anneals to the bottom strand across the bridge + SMART handle; its 3' end sits at the barcode boundary and extension reads the 12-bp cell barcode (bases 1-12) then the 8-bp UMI (bases 13-20).",
"diagram": " 5'-GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC----------------------->\n5'-...GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTACNNNNNNNNNNNNNNNNNNNNTTTTTTTT...[cDNA]-3'\n3'-...CGGACAGGCGCCTTCGTCACCATAGTTGCGTCTCATGNNNNNNNNNNNNNNNNNNNNAAAAAAAA...[cDNA]-5'\n \\___cell BC (12)___/\\_UMI(8)_/"
},
{
"read": "Index 1 (i7 sample index)",
"primer": "Nextera i7 index sequencing primer (anneals to the s7 handle: 5'-...CCGAGCCCACGAGAC-3'); only when multiplexing",
"template": "bottom",
"cycles": 8,
"note": "The index primer matches the top-strand s7 handle, so it anneals to the bottom strand with its 3' end at the index boundary and reads the 8-bp i7 sample index installed by the Nextera N70X primer.",
"diagram": " 5'-CCGAGCCCACGAGAC-------->\n5'- ...CCGAGCCCACGAGACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTG -3'\n3'- ...GGCTCGGGTGCTCTGNNNNNNNNTAGAGCATACGGCAGAAGACGAAC -5'\n reads 8-bp i7 index -->"
},
{
"read": "Read 2 (cDNA)",
"primer": "Nextera Read 2 / s7 primer (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG)",
"template": "top",
"cycles": 50,
"note": "The s7 Read 2 primer anneals to the top strand across the Nextera mosaic-end + s7 region; its 3' end points into the insert and extension reads back through the 3' cDNA fragment (50 bp; 60 bp in the retina experiment).",
"diagram": " 3'-GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-5'\n5'- ...(dT)30-[cDNA]-CTGTCTCTTATACACATCTCCGAGCCCACGAGAC-[i7]...-3'\n3'- ...(dA)30-[cDNA]-GACAGAGAATATGTGTAGAGGCTCGGGTGCTCTG-[i7]...-5'\n <-----------[reads cDNA insert]"
}
],
"whitelists": {},
"build": {
"builder_version": "llm-generic-1.0",
"deterministic": false,
"source_html_sha256": null,
"extraction_method": "claude_llm_generic",
"model": "claude-opus-4-8"
},
"title": "Drop-seq",
"description": "Drop-seq is a droplet-based, massively parallel 3\u2032 single-cell RNA-seq method. Individual cells are co-encapsulated with a DNA-barcoded microparticle (\"bead\") in a nanoliter (~1 nL, 125 \u00b5m) aqueous droplet formed by a co-flow microfluidic device; the cell lyses inside the droplet and its polyadenylated mRNAs hybridize to the bead's oligo-dT primers. Each bead carries a common SMART/PCR handle, a 12-bp split-pool cell barcode (one of 4^12 = 16,777,216), an 8-bp UMI, and a 30-bp oligo-dT. After bulk reverse transcription with template switching (STAMP formation), exonuclease treatment, whole-transcriptome PCR, and Nextera XT tagmentation of the 3\u2032 ends, the library is sequenced on Illumina: Read 1 (20 bp) yields the cell barcode + UMI, and Read 2 the cDNA.",
"reference": {
"kind": "paper",
"label": "Drop-seq Laboratory Protocol v1.1",
"path": null,
"url": "https://www.protocols.io/view/drop-seq-laboratory-protocol-mkbc4sn",
"doi": "10.17504/protocols.io.mkbc4sn"
},
"publication": {
"year": 2015,
"original_publication": {
"title": "Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets",
"journal": "Cell",
"doi": "10.1016/j.cell.2015.05.002",
"url": "http://dx.doi.org/10.1016/j.cell.2015.05.002"
},
"authors": [
{
"name": "Evan Z. Macosko",
"corresponding": true,
"email": "emacosko@genetics.med.harvard.edu",
"affiliation": "Department of Genetics, Harvard Medical School; Stanley Center for Psychiatric Research and Program in Medical and Population Genetics, Broad Institute of Harvard and MIT"
},
{
"name": "Anindita Basu",
"affiliation": "Klarman Cell Observatory, Broad Institute; School of Engineering and Applied Sciences, Harvard University"
},
{
"name": "Rahul Satija",
"affiliation": "Klarman Cell Observatory, Broad Institute; New York Genome Center; Department of Biology, New York University"
},
{
"name": "James Nemesh",
"affiliation": "Department of Genetics, Harvard Medical School; Broad Institute"
},
{
"name": "Karthik Shekhar",
"affiliation": "Klarman Cell Observatory, Broad Institute"
},
{
"name": "Melissa Goldman",
"affiliation": "Department of Genetics, Harvard Medical School; Stanley Center for Psychiatric Research, Broad Institute"
},
{
"name": "Itay Tirosh",
"affiliation": "Klarman Cell Observatory, Broad Institute"
},
{
"name": "Allison R. Bialas",
"affiliation": "The Program in Cellular and Molecular Medicine, Children's Hospital Boston"
},
{
"name": "Nolan Kamitaki",
"affiliation": "Department of Genetics, Harvard Medical School; Broad Institute"
},
{
"name": "Emily M. Martersteck",
"affiliation": "Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University"
},
{
"name": "John J. Trombetta",
"affiliation": "Klarman Cell Observatory, Broad Institute"
},
{
"name": "David A. Weitz",
"affiliation": "School of Engineering and Applied Sciences and Department of Physics, Harvard University"
},
{
"name": "Joshua R. Sanes",
"affiliation": "Department of Molecular and Cellular Biology and Center for Brain Science, Harvard University"
},
{
"name": "Alex K. Shalek",
"affiliation": "Klarman Cell Observatory, Broad Institute; Ragon Institute of MGH, MIT, and Harvard; Institute for Medical Engineering and Science and Department of Chemistry, MIT"
},
{
"name": "Aviv Regev",
"affiliation": "Klarman Cell Observatory and Department of Biology, MIT; Howard Hughes Medical Institute"
},
{
"name": "Steven A. McCarroll",
"corresponding": true,
"email": "mccarroll@genetics.med.harvard.edu",
"affiliation": "Department of Genetics, Harvard Medical School; Stanley Center for Psychiatric Research and Program in Medical and Population Genetics, Broad Institute of Harvard and MIT"
}
],
"throughput": {
"summary": "~10,000 single-cell libraries prepared per hour at 100 cells/\u00b5L (12 hr for 10,000 cells); ~1,200/hr at 12.5 cells/\u00b5L. ~6.5 cents per cell. 44,808 mouse retinal cells profiled into 39 clusters.",
"cells": "10,000 single-cell libraries in ~12 hr; 44,808 retinal cells analyzed (49,300 STAMPs sequenced)",
"rna": "At saturating depth ~44,295 transcripts / 6,722 genes per HEK cell and ~26,044 transcripts / 5,663 genes per 3T3 cell; ~12.8% transcript capture efficiency (ERCC); ~10.7% by ddPCR",
"dna": "Amplified cDNA ~1300-2000 bp; final tagmented library ~450-680 bp; yield 10-30 nM (HEK/3T3)"
},
"statistical_model": "Digital transcript counting via UMIs with edit-distance-1 (substitution-only recommended) collapse; single-cell purity and doublet rates estimated from species-mixing (binomial co-encapsulation) experiments.",
"other": [
{
"label": "Primers per bead",
"value": ">10^8 barcoded primer sites per microparticle"
},
{
"label": "Droplet size",
"value": "~125 \u00b5m diameter, ~1 nL, generated on a PDMS co-flow device (>100,000 droplets/min)"
},
{
"label": "Bead synthesis",
"value": "Reverse-direction (5'->3') phosphoramidite synthesis on Toyopearl HW-65S resin by Chemgenes"
},
{
"label": "Capture efficiency",
"value": "~12.8% (ERCC spike-in), corroborated at ~10.7% by droplet digital PCR"
},
{
"label": "Accession",
"value": "GEO: GSE63473"
},
{
"label": "Protocol source",
"value": "Drop-seq Laboratory Protocol v1.1 (Aug 6, 2015), Macosko & Goldman, McCarroll Lab (www.mccarrolllab.com/dropseq)"
}
]
},
"modality": "RNA",
"method_type": "droplet",
"data_processing": {
"summary": "Read 1 supplies the 12-bp cell barcode + 8-bp UMI, which are tagged onto the paired cDNA read; the cDNA (Read 2) is aligned to the reference genome, reads are grouped by cell barcode, and UMIs are collapsed per gene to give digital transcript counts \u2014 producing a cell-by-gene digital gene-expression (DGE) matrix that is then clustered. The paper and protocol point to the McCarroll-lab 'Drop-seq tools' software (Nemesh, with help from Wysoker) and an accompanying downloadable informatics guide.",
"stages": [
{
"id": "prep",
"label": "Barcode Tagging & Trimming"
},
{
"id": "align",
"label": "Alignment"
},
{
"id": "quant",
"label": "Quantification"
},
{
"id": "analysis",
"label": "Analysis"
}
],
"nodes": [
{
"id": "tag_bc",
"label": "Tag reads with barcode + UMI",
"tool": "Drop-seq tools",
"stage": "prep",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "trim_adapter",
"label": "Trim SMART adapter",
"tool": "Drop-seq tools",
"stage": "prep",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "trim_polya",
"label": "Trim poly(A) tail",
"tool": "Drop-seq tools",
"stage": "prep",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "align",
"label": "Align cDNA to genome",
"tool": "STAR",
"stage": "align",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "group_bc",
"label": "Group reads by cell barcode",
"tool": "Drop-seq tools",
"stage": "quant",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "collapse_umi",
"label": "Collapse UMIs (edit distance 1)",
"tool": "Drop-seq tools",
"stage": "quant",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "build_dge",
"label": "Build DGE matrix",
"tool": "Drop-seq tools",
"stage": "quant",
"scope": "bulk",
"terminal": false,
"viz_only": false
},
{
"id": "cluster",
"label": "Cluster cells",
"tool": "",
"stage": "analysis",
"scope": "bulk",
"terminal": true,
"viz_only": false
},
{
"id": "species_mix",
"label": "Estimate doublet rate",
"tool": "",
"stage": "analysis",
"scope": "bulk",
"terminal": true,
"viz_only": false
}
],
"edges": [
{
"from": "tag_bc",
"to": "trim_adapter",
"kind": "sequential"
},
{
"from": "trim_adapter",
"to": "trim_polya",
"kind": "sequential"
},
{
"from": "trim_polya",
"to": "align",
"kind": "sequential"
},
{
"from": "align",
"to": "group_bc",
"kind": "sequential"
},
{
"from": "group_bc",
"to": "collapse_umi",
"kind": "sequential"
},
{
"from": "collapse_umi",
"to": "build_dge",
"kind": "sequential"
},
{
"from": "build_dge",
"to": "cluster",
"kind": "branch"
},
{
"from": "build_dge",
"to": "species_mix",
"kind": "branch"
}
],
"statistical_model": "Digital transcript counting via UMIs collapsed at edit distance 1 (substitution-only); single-cell purity and doublet rate estimated from species-mixing (binomial co-encapsulation) analysis."
}
}
|