GSM5738152: 15d organoids, ctr rep2; Homo sapiens; RNA-Seq
No RNA extraction was performed for the scRNA-seq data generation (see library construction protocol) Cells were encapsulated together with barcoded microparticles (Macosko- 2011-10 V+, ChemGenes) using the Dolomite Bio Nadia instrument, using the standard manufacturer's dropseq-based scRNA-seq protocol. Droplets were broken immediately after collection and cDNA libraries generated as previously described (Wyler et al., 2021). First strand cDNA was amplified by equally distributing beads from one run to 24 Smart PCR reactions (50 μl volume; 4 + 9 to 11 cycles). 20 μl fractions of each PCR reaction were pooled, then double-purified with 0.6 volumes of AMPure XP beads. Amplified cDNA libraries were assessed and quantified on a BioAnalyzer High Sensitivity Chip (Agilent) and the Qubit dsDNA HS Assay. Nine-hundred pg of each cDNA library was fragmented, amplified (13 cycles) and indexed for sequencing with the Nextera XT v2 DNA sample preparation kit (Illumina) using custom primers enabling 3'-targeted amplification. The libraries were purified with AMPure XP Beads, quantified and sequenced on Illumina sequencers (first run: concentration 2pM; Hiseq 3000/4000 SBS kit (150 cycles) paired-end mode; read 1=75 using the custom primer Read1custseqB (8bp) read 2=75. Second run: concentration 2 pM; NextSeq 500/550 High Output v2 kit (75 cycles) in paired-end mode; read 1 = 21 bp using the custom primer Read1CustSeqB (8 bp) read 2 = 63 bp). Single-cell RNA-seq (Drop-seq)