title: GSM3498622: Murine_Hep_Orgs_sc; Mus musculus; RNA-Seq
library_construction_protocol: Hepatocytes were isolated by two steps of collagenase perfusion. Organoids were withdrawn from Matrigel. For bulk sequencing, mRNA were isolated by trizol method according to manufacter's protocol. RNA-sequencing libraries were prepared based on the DESeq2 method (Hashimshony et al., 2016; Love et al., 2014). For single cell sequencing, Organoids were digested by triple into single cells. Live positive single cells were sorted in 384-well hard shell plates (Biorad) with 5 μl of vapor-lock (QIAGEN) containing 100 nl of RT primers (consisting of a 24 bp polyT stretch, a 8bp random molecular barcode (UMI), a cell-specific barcode, the 5'Illumina TruSeq small RNA kit adaptor and a T7 promoter), dNTPs and mRNA ERCC Spike-Ins (Agilent) and immediately frozen to −80°C. Before starting with SORT-seq, cells were first lysed 5 min at 65°C. RNA was processed using the previously described SORT-seq tecnique, which is an implemented version of the CEL-seq2 technique. Libraries were sequenced on an Illumina NextSeq500 using 75bp paired end sequencing.