Datasets:
sequencing
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scg-protocols-v1

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Molecular event:
Unmethylated lambda DNA is added as a bisulfite-conversion control. MspI digests genomic DNA at CCGG sites, generating reduced-representation genomic fragments enriched for CpG-rich regions.
Product structure:
MspI-digested genomic DNA fragments
Step 3. End repair and dA-tailing
Input substrate:
* MspI-digested genomic DNA fragments
Added oligos/reagents:
* Klenow fragment, exo-
* dATP
* dCTP
* dGTP
* Tango buffer
Molecular event:
Digested DNA fragments are end-repaired and dA-tailed to prepare them for ligation to TruSeq adapters.
Product structure:
* end-repaired, dA-tailed MspI genomic fragments
Step 4. Adapter ligation
Input substrate:
* end-repaired, dA-tailed MspI genomic fragments
Added oligos/reagents:
* premethylated indexed TruSeq adapters
* T4 DNA ligase
* ATP
* Tango buffer
Molecular event:
Premethylated indexed TruSeq adapters are ligated to the dA-tailed DNA fragments. The indexed adapter contains a 6-base sample index. The adapter cytosines are methylated so that they are protected during bisulfite conversion.
Product structure:
TruSeq universal adapter
* MspI genomic DNA fragment
* indexed TruSeq adapter
Step 5. Bisulfite conversion
Input substrate:
* adapter-ligated genomic DNA fragments
Added oligos/reagents:
* bisulfite conversion reagent
* tRNA carrier during purification
Molecular event:
Adapter-ligated DNA is bisulfite converted. Unmethylated cytosines in genomic DNA are converted, while methylated cytosines remain protected. The converted DNA is then purified after bisulfite treatment.
Product structure:
* bisulfite-converted adapter-ligated DNA fragments
Step 6. First-round PCR enrichment
Input substrate:
* purified bisulfite-converted adapter-ligated DNA
Added oligos/reagents:
* QP1 primer
* QP2 primer
* PfuTurbo Cx hotstart DNA polymerase
* dNTP mix
Molecular event:
The bisulfite-converted library molecules are amplified using QP1 and QP2 primers. PfuTurbo Cx is used because it is resistant to uracil stalling.
Product structure:
* first-round PCR-amplified scRRBS library molecules
Step 7. Second-round PCR enrichment
Input substrate:
* first-round PCR product
Added oligos/reagents:
* QP1 primer
* QP2 primer