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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