GSM1971277: DNMT_B8; Mus musculus; Bisulfite-Seq
Cells were sorted into 2.5ul RLT plus lysis buffer (QIAGEN) containing SUPERase-In (Ambion). scBS-seq libraries were prepared as previously described (Smallwood et al., Nature Methods 11, 817-820) but with the following modifications: bisulfite conversion was carried out using Imprint reagent (Sigma, MOD50), with desulphonation and purification using magnetic beads, following the manufacturers’ instructions but with half volumes. Primers for first and second strand synthesis contained a 3’ random hexamer instead of a nonamer and biotin capture of first strand synthesis products was omitted, substituting an extra AMPure XP (Beckman Coulter, A63881) purification between second strand synthesis and PCR amplification. In order to avoid potential batch effects which could mask subtle differences between samples all libraries were prepared in parallel with the pre-PCR purification steps carried out using a Bravo Workstation pipetting robot (Agilent Technologies).