GSM5532202: PHH_1336; Homo sapiens; RNA-Seq
For single cell sorting, cells were washed one time with cold PBS containing 1% BSA, and then sorted using BDFACSMelody with a 100 µm nozzle into a 384-well plate containing 100 nl unique Cel-seq primer solution and 5 µl of mineral oil (Sigma, M5310). Single-cell RNA libraries were constructed according to the mCEL-seq2 protocol . Briefly, 100 nL lysis buffer (0.2% Triton X-100, 2mM dNTPs, 1:50.000 ERCC ExFold RNA spike-in (Thermo, 4456739)) was added per well using the Nanodrop Ns-2 Stage (BioNex). For the in vitro reverse transcription reaction, SuperScript™ II Reverse Transcriptase (Invitrogen, 18064-14) was used with the following the protocol, 4 ⁰C for 5 min; 25 ⁰C for 10 min; 42 ⁰C for 60 min; 70 ⁰C for 10 min. Next, 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 added per well for second-strand synthesis following the protocol, 16 ⁰C for 2 hrs. All the cDNA from one 384-well plate was pooled. In vitro transcription was performed using the MEGAscript™ T7 Transcription Kit (Invitrogen, AMB 1334-5) by incubating the reaction at 37 ⁰C for 14 hrs. The amplified RNA was fragmented and used for cDNA synthesis using Superscript II and the random octamer primer. Libraries were amplified for 9 cycles for the hepatocyte libraries and 8 cycles for the liver organoid libraries using Phusion® High-Fidelity PCR Master Mix (NEB, M0531S) with the Nextflex primers, and quantified on the bio-analyzer using DNA high-sensitivity kit (Agilent, 5067-4626). Barcoded libraries were sequenced on an Illumina NextSeq 500 with 38bp paired-end reads at a depth of about 25 million reads per plates.