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TABLE 1 | Oligo sequences of adapters. |
Oligo name Sequence (all sequences read 5'-3') |
TruSeq universal adapter AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC*T |
TruSeq adapter (index 1) GATCGGAAGAGCACACGTCTGAACTCCAGTCACATCACGATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 2) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCGATGTATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 3) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 4) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTGACCAATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 5) GATCGGAAGAGCACACGTCTGAACTCCAGTCACACAGTGATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 6) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGCCAATATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 7) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCAGATCATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 8) GATCGGAAGAGCACACGTCTGAACTCCAGTCACACTTGAATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 9) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGATCAGATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 10) GATCGGAAGAGCACACGTCTGAACTCCAGTCACTAGCTTATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 11) GATCGGAAGAGCACACGTCTGAACTCCAGTCACGGCTACATCTCGTATGCCGTCTTCTGCTTG |
TruSeq adapter (index 12) GATCGGAAGAGCACACGTCTGAACTCCAGTCACCTTGTAATCTCGTATGCCGTCTTCTGCTTG |
Index sequences are six bases, underlined. |
• DNA loading buffer (6×; Clontech, cat. no. 9156) |
• GeneRuler low-range DNA ladder (ready-to-use; Thermo Scientific, cat. no. SM1193) |
• DTT (Thermo Scientific, cat. no. R0861) |
• KAPA HiFi HotStart ReadyMix (2×; Kapa Biosystems, cat. no. KK2602) EQUIPMENT |
• PCR tubes, 0.5-ml thin-walled with flat cap (Axygen, cat. no. PCR-05-C) |
• PCR tubes, 0.2-ml thin-walled with flat cap (Axygen, cat. no. PCR-02-C) |
• 1,000-μl universal fit filter tips (Axygen, cat. no. TF-1000-R-S) |
• 200-μl universal fit filter tips (Axygen, cat. no. TF-200-R-S) |
• 100-μl universal fit filter tips (Axygen, cat. no. TF-100-R-S) |
• 10-μl universal fit filter tips (Axygen, cat. no. TF-300-R-S) |
• DNA LoBind tubes, 1.5 ml (Eppendorf, cat. no. 022431021) |
• DNA LoBind tubes, 2.0 ml (Eppendorf, cat. no. 022431048) |
• Cell culture-treated dishes (60 mm; Thermo Scientific, cat. no. 130181) |
• Falcon conical centrifuge tubes (15 ml; Corning Life Sciences, cat. no. 14-959-49D) |
• Magnetic rack (Diagenode, cat. no. kch-816-001) |
• 10 μM filter spin (Zymo, cat. no. C1007-50) |
• Dark Reader transilluminator (Clare Chemical Research, cat. no. DR88X) |
• Flaming/Brown micropipette puller (Sutter Instrument, P-1000) |
• Refrigerated microcentrifuge (5415R; Eppendorf, cat. no. 022621408) |
• Nonrefrigerated microcentrifuge (Heraeus Pico17; Thermo Scientific, cat. no. 75002410) |
• Thermomixer (Eppendorf, cat. no. 5355 000.011) |
• Qubit 2.0 fluorometer (Thermo Scientific, cat. no. Q32866) |
• Borosilicate glass capillary (Sutter Instrument, cat. no. B100-58-10) ! CAUTION Be careful with the glass capillary; handle it with appropriate protection. |
• Aspirator tube assemblies for calibrated microcapillary pipettes (Sigma-Aldrich, cat. no. A5177) |
• HL-2000 HybriLinker (UVP, cat. no. 95-0031-02) |
• Vortex (Scientific industries, cat. no. SI-0246) |
• Thermocycler with a 96-well block (TProfessional Gradient 96; Biometra, cat. no. 846-070-801) |
• 16-tube magnetic rack (Diagenode, cat. no. B04000001 (kch-816-001)) |
• ChemiDoc XRS+ System with Image Lab Software (computer-connected, Bio-Rad, cat. no. 170-8265) |
• 4-gel vertical electrophoresis system (Bio-Rad, cat. no.165-8006) |
• PowerPac basic power supply (Bio-Rad, cat. no. 164-5050) |
• Transference decoloring shaker (Qilinbeier, cat. no. TS-8) |
• Fragment Analyzer (Advanced Analytical Technologies, cat. no. FA12) |
## Software |
• Perl. Download freely from http://www.perl.org/ |
• Trim_galore (version 0.3.7). Freely available at http://www.bioinformatics. babraham.ac.uk/projects/trim_galore/ |
• Bowtie (version 1, ref. 38). Download from http://bowtie-bio.sourceforge. net/index.shtml |
• Bismark (version 0.7.6, ref. 35). Download from http://www.bioinformatics. babraham.ac.uk/projects/bismark/. Other similar tools can also be used |
• Picard toolkit. Download from http://broadinstitute.github.io/picard/. Other similar tools can also be used |
• Samtools39. Download from http://samtools.sourceforge.net/ |
• Java. Download from https://www.java.com/ |
• Custom scripts. An archive of all customized scripts used in this protocol is available as Supplementary Data. Refer to Table 2 for details of each script REAGENT SETUP |
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