Instrument: Illumina HiSeq 2500
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: PAIRED
Construction protocol: The released genomic DNA were used to construct the PBAT library, as previously described (Guo et al., 2015; Smallwood et al., 2014). Briefly, the single-cell genomic DNA, together with unmethylated lambda DNA (New England Biolabs) spike-ins, were subjected to bisulfite conversion using a MethylCode Bisulfite Conversion Kit (Invitrogen) according to the manufacturer’s instructions. The bisulfite converted DNAs were then annealed using random nonamer primers with a 5’- biotin tag and a truncated Illumina P5 adaptor (5’-biotin- CTACACGACGCTCTTCCGATCTNNNNNNNNN-3’) supplemented with 50 units of klenow polymerase (3’ to 5’ exo-, New England Biolabs). The excess primers were removed using 40 U exonuclease I (NEB) before DNA was purified using 0.8× Agencourt Ampure XP beads (Beckman Coulter). Then, the newly synthesized DNA strands were immobilized using the Dynabeads M280 Streptavidin (Invitrogen), and the original bisulfite-treated DNA templates were removed via two rounds of 0.1 N NaOH washes. The second strands were synthesized using 50 units klenow polymerase with random nonamer primers containing a truncated P7 Illumina adaptor (5’-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3’). The beads were further collected and washed several times, and the library was generated with 13 cycles of PCR amplifications using 1 U Kapa HiFi HS DNA Polymerase (Kapa Biosystems), together with 0.4 μM Illumina Forward PE1.0 primer (5’-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’) and 0.4 μM pre-indexed Illumina Reverse primer (5’-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3’; the underlined hexamer indicates the index sequences). Amplified libraries were purified with 0.8× Agencourt Ampure XP beads twice and were assessed on the Agilent Bioanalyzer 2100 platform and quantified with a standard curve-based qPCR assay (Kapa Biosystems). The final quality- ensured libraries were pooled and sequenced on the Illumina HiSeq2500 sequencer for 150 bp paired-end sequencing.