Illumina HiSeq 2500 paired end sequencing; MERVL/Zscan4 network activation results in transient genome-wide DNA demethylation of mESCs
MERVL/Zscan4 network activation results in transient genome-wide DNA demethylation of mESCs
E14 ESCs were cultured in DMEM containing 15% fetal calf serum and 103 U/mL leukemia inhibitory factor (LIF); for 2i/LIF experiments, cells were cultured in serum-free N2B27 supplemented with LIF, 1 μM PD0325901, and 3 μM CHIR99021 inhibitors. The Zscan4c::emerald plasmid (90636) was kindly provided by Minoru Ko (Zalzman et al., 2010), and the MERVL::tdTomato reporter (Macfarlan et al., 2012) was a gift from Samuel Pfaff (Addgene #40281). Reporter stable clonal lines were generated by transfection (Fugene6, Promega), drug selection, and subcloning. Flow cytometry analysis was performed using the BD LSR Fortessa and sorts performed on the BD Aria III or BD Influx High-Speed Cell Sorter. We sorted four 96-well plates where each plate had five populations: 56 cells in a Zscan4+MERVL+ population, 16 cells in a Zscan4+ only population, 8 cells in a negative population with slight MERVL expression, 14 cells from a completely negative populations, and 2 empty wells for control. To attempt to combat potential batch effects during the library preparation, wells were transferred to new plates mixing rows from different sorting plates in to same library plate. Single-cells were sorted in 2uL of Lysis Buffer (1:20 solution of RNase Inhibitor (Clontech, cat. no. 2313A or Invitrogen RNAse OUT) in 0.2% v/v Triton X-100 (Sigma-Aldrich, cat. no. T9284) in 96 well plates, spun down and immediately frozen at -80 degrees. Oligo-dT primer, dNTPs (ThermoFisher, cat. no. 10319879) and ERCC RNA Spike-In Mix (1:25,000,000 final dilution, Ambion, cat. no. 4456740) were then added, and Reverse Transcription and PCR were performed as in (Picelli et al., 2014). The cDNA libraries for sequencing were prepared using Nextera XT DNA Sample Preparation Kit (Illumina, cat. no. FC-131-1096), according to the protocol supplied by Fluidigm (PN 100-5950 B1). Libraries from 96 single cells were pooled and purified using AMPure XP beads (Beckman Coulter)