Instrument: Illumina HiSeq 3000
Strategy: OTHER
Source: GENOMIC
Selection: other
Layout: SINGLE
Construction protocol: The DNAs were end-repaired using the End-It™ DNA End-Repair Kit (Lucigen, Radnor, PA, USA). The reaction was done in 10 µL reaction volume according to the manufacturer's instruction. The reaction was terminated by adding 115 µL 10 mM Tris-Cl buffer (pH7.5) containing 1 mM EDTA, and the DNAs were extracted by the phenol-chloroform extraction, followed by the ethanol precipitation as described above. The precipitate is re-suspended in 5 µL Qiagen Buffer EB (10 mM Tris-Cl buffer (pH 8.5)). The addition of 3' A overhangs to the end-repaired DNA fragments was done using the Klenow fragment (3'→5' exo-) (New England BioLabs, Ipswich, MA, USA) and 1 mM deoxyadenosine triphosphate in the reaction buffer. The DNA fragments were incubated at 37°C for 20 min and the enzymes were inactivated at 75°C for 20 min. The Illumina Adaptor Oligo Mix (Illumina, San Diego, CA, USA) was ligated to the 3' A overhangs of the DNA fragments using the T4 DNA ligase (New England BioLabs) by incubating at room temperature for 3 hr. The DNA fragment with the adaptors bound was PCR amplified using the Phusion High-Fidelity PCR Master Mix (New England BioLabs) and the 125 nM PE PCR Primer 1.0 (Illumina). The PCR was done under the following condition: 98°C for 10 min, followed by a cycle of 30 sec at 68°C and 30 sec at 72°C (repeat 18 cycles in the case of 3,000 cells or 25 cycles in the case of a single cell). The 2% [w/v] agarose gel electrophoresis was done to isolate the 140-350 bp fragments and purify using the QIAquick Gel Extraction kit (Qiagen). The concentration of the purified DNAs was measured using Qubit dsDNA HS kit (Thermo Fisher Scientific). The paired-end sequencings were run using the Illumina MiSeq and HiSeq2000.