alias: GSM4270863
title: GSM4270863: s.NJA01303.I05; Mus musculus; RNA-Seq
library_construction_protocol: Cells were stained with FITC anti-CD326 and 7AAD (Sony Biotechnology) in order to monitor number of epithelial cells and select live cells. Additionally, paneth cells were enriched using Brilliant Violent anti-CD24. Single cells were distinguished from multiplets using FSC-H and FSC-W gating and the gating scheme was visually confirmed via phase contrast microscopy (Fig. 1a). Cells were sorted into 384- or 96-well plates containing hypotonic lysis buffer using a SH800S FACS sorter (Sony). Plates were sealed immediately after sorting with microseal F foil (Biorad) and centrifuged at 4000g at 4°C for 5 min before being frozen on dry ice and stored at -80°C. Single-cell and multiplet RNA-Seq libraries were generated as described. Briefly, single-cells collected in 384-well plates were lysed, followed by reverse transcription with template-switch using an LNA-modified template switch oligo to generate cDNA. After 21 cycles of pre-amplification, DNA was purified and analysed on an automated Fragment Analyzer (Advanced Analytical). Each cell's cDNA fragment profile was individually inspected and only wells with successful amplification products (concentration higher than 0.06 ng/ul) and with no detectable RNA degradation were selected for final library preparation. Tagmentation assays and barcoded sequencing libraries were prepared using Nextera XT kit (Illumina) according to the manufacturer's instructions. Barcoded libraries were pooled and subjected to 75 bp paired-end sequencing on the Illumina NextSeq 500 instrument.
identifiers.namespace: GEO
identifiers.id: GSM4270863