GSM7790628: naïve SpiB+ epithelila cells, scRNAseq, rep4; Mus musculus; RNA-Seq
Single-cell (sc)-RNA-seq using a modified CEL-Seq2 protocol. Cell were sorted into into a chilled 384-well PCR plate (Greiner, 785290) containing 1.2μl of primer/lysis mix [20 nM indexed polydT primer (custom made, IDT), 1:6.000.000 dilution of ERCC RNA spike-in mix (Ambion - 4456740), 1 mM dNTPs (NEB - N0446S), 1.2 units SUPERaseIN Rnase Inhibitor (Thermo Fisher - AM2696), 0.2 % Triton X-100 solution (Sigma Aldrich – 93443-100ml), DEPC water (Thermo Fisher - AM9920)] Single cell transcriptome libraries were generated by adapting the CelsSeq2 protocol [Hashimshony et al., 2016] as follows: Samples were pooled after first strand cDNA synthesis, treated with Exonuclease 1 for 30 minutes, followed by a 1.2X bead clean-up. Second strand synthesis was performed using NEBNext Second Strand Synthesis module (NEB #E6111S) in a final reaction volume of 20 ul and NucleoMag NGS Clean-up and Size select magnetic beads (Macherey-Nagel - 7449970.5) were used for all DNA purification and size selection steps.]