GSM2299711: RRBS_40_45B; Mus musculus; Bisulfite-Seq
Extracted DNA was spiked with a small amount of lambda DNA (0.05pg per 6ng of genomic DNA) for bisulphite conversion control, digested with MspI endonuclease (Fermentas), end-repaired (Klenow fragment exo-, Fermentas, with 10nM dATP, 1nM dCTP and 1nM dGTP) and ligated with 5mC-adaptors (Illumina) with T4 ligase (Fermentas).  Bisulphite conversion was done using the EZ DNA Methylation-Direct Kit (Zymo Research) and DNA was amplified by 18 cycles of PCR using Pfu Turbo Cx polymerase (Stratagene).  DNA was purified using SPRI beads (Agencourt). RNA was extracted using TRIsure reagent (Bioline) followed by RNA Clean & Concentrator (Zymo Research) with on-column DNase treatment (RNasefree DNase I, Life Technologies). For RNA-seq libraries:  Reverse transcription was performed using SuperScript III (Life Technologies), followed by second DNA strand synthesis using dUTPs instead of dTTPs and DNA polymerase I (NEB); libraries were constructed using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB), including dUTP excision step by USER Enzyme (NEB) before PCR. RRBS: Extracted DNA was spiked with a small amount of lambda DNA (0.05pg per 6ng of genomic DNA) for bisulphite conversion control, digested with MspI endonuclease (Fermentas), end-repaired (Klenow fragment exo-, Fermentas, with 10nM dATP, 1nM dCTP and 1nM dGTP) and ligated with 5mC-adaptors (Illumina) with T4 ligase (Fermentas).  Bisulphite conversion was done using the EZ DNA Methylation-Direct Kit (Zymo Research) and DNA was amplified by 18 cycles of PCR using Pfu Turbo Cx polymerase (Stratagene).  DNA was purified using SPRI beads (Agencourt).  For PBAT: As in Stewart et al. (2015): Cells were lysed in EB buffer (Qiagen) with 0.5% SDS (Sigma, 161-0418) and bisulfite-treated using theone-stepmodification procedure in the Imprint DNA modification kit(Sigma,MOD50-1KT).The resulting DNA was purified using the EZ DNA methylation direct kit (Zymo, D5020). First strand synthesis was performed using Klenow Exo− (New England Biolabs, M0212S) and a custom streptavidin-conjugated adaptor containing standard Illumina adaptor sequences and 9 base pairs (bp) of random sequence (9N). This was followed by exonuclease I treatment (New England Biolabs, M0293S), purification using SPRI beads (1.8× ratio; Fisher Scientific, 09-981123), and binding to biotin beads (Life Technologies, 11205D). Samples were then subjected to second strand synthesis, again using custom primer, followed by 10 cycles of library amplification using Phusion polymerase (Thermo Scientific, F-530S) before final purification using SPRI beads.