GSM8290422
RNA was extracted by 2x heat lysis (65C) in 384 well, which were immediately used for downstream single cell RNA sequencing reactions (CELseq2) Single-cell RNA sequencing was conducted using CEL-seq2 at Single Cell Discoveries (Utrecht, NL), following the protocols by Hashimshony et al., 2016 and Muraro et al., 2016. The plates were preloaded with barcoded poly-T primers designed to specifically amplify mRNA and introduce a 6bp unique molecular identifier (UMI), an 8bp cell barcode, and a T7 RNA polymerase binding site. Additionally, ERCC spike-ins were preloaded into the plates to enable quality control of reactions in individual wells. Cells were lysed by heating at 65°C for 5 minutes, followed by rapid chilling on ice for 2 minutes, repeated twice. Superscript II (ThermoFisher Scientific) was used for reverse transcription of mRNA, which was then converted into second-stranded DNA by DNA polymerase I (ThermoFisher Scientific). The material from each plate was pooled, cleaned using Ampure beads (Beckman Coulter, #A63881), and amplified by in vitro transcription overnight with the MEGAscript T7 transcription kit (ThermoFisher Scientific, #AMB1334). After a second round of cleanup, the amplified RNA was measured with Agilent RNA 6000 pico chips (Agilent #5067-1513), reverse-transcribed using random hexamer primers to introduce Truseq Small RNA kit RP1 primer binding sites (Illumina), and finally converted into DNA libraries with custom rpi primers adapted from the Truseq Small RNA kit (Illumina)106. After two rounds of Ampure bead cleanup and quality control using the Agilent High Sensitivity DNA Kit (5067-4626), the libraries were sent for paired-end sequencing (26bp for read 1 and 50bp for read 2) on the Nextseq500 at the Utrecht Sequencing Facility (USEQ, Life Sciences faculty, Utrecht University). Library was performed according to manufacturer's instructions. Illumina sequencing libraries were prepared with the TruSeq small RNA primers (Illumina) and sequenced paired-end at 75 bp read length the Illumina NextSeq.