GSM4655705: GC071392_TCTTACGC-GTAGAGGA; Mus musculus; RNA-Seq
For the isolation of Day 0 time point of single-cell transcriptome analysis, 96 Xi-GFP cells were sorted into a 96-well multiwell plate with 2 µl lysis buffer, followed by initial steps of smart-seq2 protocol (see below). Remaining cells were plated for reprogramming directly after sorting, 50.000 cells per one well of a 12 well plate. Reprogramming was induced by doxycycline (2 μg/ml final) in mouse ESC medium in the presence of ascorbic acid (50 μg/ml final). The medium together with doxycycline and ascorbic acid was replaced every two days and maintained throughout entire reprogramming experiments. To derive iPSCs from picked colonies, the same conditions were used with dox and AA withdrawal after picking. Next, FUT4+ reprogramming intermediates were sorted into single wells, each with 2 µl of the lysis buffer [0,2% Triton X100, 1:20 RRI (Recombinant RNase Inhibitor Takara)] and snap-frozen in -80°C until cells from all timepoints were collected. All samples were processed following the smart-seq2 protocol (Picelli et al). cDNA synthesis was done starting from 500 pg of input RNA, followed by library preparation from 80 pg of cDNA using the Nextera XT kit (Illumina, FC-131-1096). Indexing was performed with the Nextera XT index Kit V2 (Illumina, FC-131-2003). The quality of cDNA and individual libraries was assessed using an Agilent 2100 Bioanalyzer system.