title: GSM5421291: BRCO-1060; Homo sapiens; RNA-Seq
library_construction_protocol: Organoids (ICO and BRCO lines 6, 7 and 10) were incubated with Calcein for 45 min at 37⁰C before dissociation for live staining of the cells. To get single cells, organoids were then dissociated and digested by 0.05% Trypsin-EDTA following the online protocol. Single-cells were obtained using the flow cytometer FACS Aria (BD Biosciences) equipped with a 130 µm nozzle. Only GFP positive cells (living cells) were sorted into 384-well plates containing a unique CEL-seq primer and 5 µL mineral oil (Sigma, M5310) per well. Plates were stored at -80 ⁰C until they were processed. Single-cell RNA libraries were prepared according to the mCEL-seq2 protocol. Briefly, plates were thawed on ice, and then 100 nL lysis buffer (0.2% Triton X-100, 2mM dNTPs, 1:50.000 ERCC ExFold RNA spike-in (Thermo, 4456739)) was dispensed per well using the Nanodrop Ns-2 Stage (BioNex). SuperScript™ II Reverse Transcriptase (Invitrogen, 18064-14) was used in reverse transcription reaction mix following the protocol, 4 ⁰C for 5 min; 25 ⁰C for 10 min; 42 ⁰C for 60 min; 70 ⁰C for 10 min. 960 nL second-strand reaction mix containing E. coli DNA ligase (NEB, M0205L), E. coli DNA polymerase I (NEB, M0209L) and Random hexamer (sigma, 11034731001) were used per well for second-strand synthesis with the following protocol, 16°C for 2 hours. Next, cDNA from one 384-well plate was pooled, and in vitro transcription was performed using the MEGAscript™ T7 Transcription Kit (Invitrogen, AMB 1334-5) by incubating the reaction at 37°C for 14 hours. Afterwards, the amplified RNA was fragmented and cDNA was converted using Superscript II  and the random octamer primer. Libraries were amplified for 9 cycles using Phusion® High-Fidelity PCR Master Mix (NEB, M0531S) with the Nextflex primers, and quantified on the bio-analyzer using DNA high-sensitivity kit. Barcoded libraries were sequenced on an Illumina NextSeq 500 with 38bp paired-end reads at a depth of 24.2-32 million reads per plate. Cel-seq2