title: GSM2028285: WT1_E6.5_Epiblast_PBAT; Mus musculus; OTHER
library_construction_protocol: DNA was extracted from isolated epiblasts by using the Qiagen AllPrep DNA/RNA Mini Kit. About 5 ng genomic DNA were used to construct the PBAT library using the protocol slightly modified from previously published. Briefly, the isolated genomic DNAs, together with 1% unmethylated lambda DNA (Thermo Scientific), were converted and purified with MethylCode Bisulfite Conversion kit (Invitrogen) following the manufacturer’s instructions. Then random nonamer primers with a 5’ biotin tag and a truncated Illumina P5 adaptor (5’-biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3’) and 50 units of Klenow polymerase (3’ to 5’ exo-, NEB) were used to amplify the bisulfite-converted templates linearly. The excess primers remained in last reaction were removed using 40U Exonuclease I (NEB) before amplified products were purified with 0.8 x Agencourt Ampure XP beads (Beckman Coulter). The newly synthesized DNA strands with biotin were captured with Dynabeads M280 Streptavidin (Invitrogen), and the original bisulfite-treated DNA templates were washed away by 0.1 N NaOH. 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 with double strand DNA products were further collected and washed several times, and the libraries were generated with 5-6 cycles of PCR amplifications using 1 unit 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 x Agencourt Ampure XP beads twice and were assessed with the Fragment Analyzer (Advanced analytical) and quantified with a standard curve-based qPCR assay (Roche). The final quality-ensured libraries were pooled and sequenced on the Illumina X Ten sequencer for 150 bp paired-end sequencing. whole-genome bisulfite sequencing by post-bisulfite adaptor tagging (PBAT)