E14 ESCs were cultured in serum and LIF or 2i media as described previously16 (link). Single cells were collected by FACS following ToPro-3 and Hoechst 33342 staining to select for live cells with low DNA content (i.e. G0 or G1 phase cells). Cells were collected in RLT plus lysis buffer (Qiagen) containing 1 U/μl SUPERase-In (Ambion) and processed using the G&T-seq protocol7 (link), but following physical separation of mRNA and genomic DNA from single cells, the DNA was eluted into 10 μl of H2O.
Single-cell bisulfite libraries were then prepared as previously described3 (link) but with the following modifications. Conversion was carried out using EZ Methylation Direct bisulfite reagent (Zymo) on purified DNA in the presence of AMPure XP beads (Beckman Coulter) following G&T-seq. Purification and desulphonation of converted DNA was performed with magnetic beads (Zymo) on a Bravo Workstation (Agilent), eluting into the mastermix for the first strand synthesis. Primers for first and second strand synthesis contained a 3′-random hexamer and biotin capture of first strand products was omitted, however an extra 0.8× AMPure XP purification was performed between second strand synthesis and PCR. Each pre-PCR AMPure XP purification was carried out using a Bravo Workstation. To avoid batch effects all libraries were prepared in parallel in a 96 well plate. Purified scBS-seq libraries were sequenced in pools of 16-20 per lane of an Illumina HiSeq2000 using 125-bp paired-end reads.
RNA sequencing libraries were prepared from the single-cell cDNA libraries using the Nextera XT kit (Illumina) as per the manufacturer's instructions but using one-fifth volumes. Multiplexed library pools were sequenced on one lane of an Illumina HiSeq2000 generating 125-bp paired-end reads.
Single-cell bisulfite libraries were then prepared as previously described3 (link) but with the following modifications. Conversion was carried out using EZ Methylation Direct bisulfite reagent (Zymo) on purified DNA in the presence of AMPure XP beads (Beckman Coulter) following G&T-seq. Purification and desulphonation of converted DNA was performed with magnetic beads (Zymo) on a Bravo Workstation (Agilent), eluting into the mastermix for the first strand synthesis. Primers for first and second strand synthesis contained a 3′-random hexamer and biotin capture of first strand products was omitted, however an extra 0.8× AMPure XP purification was performed between second strand synthesis and PCR. Each pre-PCR AMPure XP purification was carried out using a Bravo Workstation. To avoid batch effects all libraries were prepared in parallel in a 96 well plate. Purified scBS-seq libraries were sequenced in pools of 16-20 per lane of an Illumina HiSeq2000 using 125-bp paired-end reads.
RNA sequencing libraries were prepared from the single-cell cDNA libraries using the Nextera XT kit (Illumina) as per the manufacturer's instructions but using one-fifth volumes. Multiplexed library pools were sequenced on one lane of an Illumina HiSeq2000 generating 125-bp paired-end reads.
