GSM6066472: p6_J14_sc_HME-RAS-ER_1c [HFFTNBBXY_6_IDT-DUI-NXT-268]; Homo sapiens; RNA-Seq
Briefly, freshly harvested single cells were sorted into 96-well plates containing the lysis buffer (0.2% Triton X-100, 1U/  l RNase inhibitor, Thermo Fisher Scientific). RT was performed using SuperScript II (Thermo Fisher Scientific) in the presence of 1   M oligo-dT30VN (IDT), 1   M Template-switching oligonucleotides (QIAGEN), and 1 M betaine. Full-length scRNAseq libraries were prepared using the Smart-seq2 protocol (Picelli et al., 2013) with minor modifications. cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Roche) and IS PCR primer (IDT), with 23 cycles of amplification. Following purification with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with Nextera XT indexes (Illumina). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sequencing of Nextera libraries was carried out using a HiSeq4000 (Illumina) to an average sequencing depth of 0.5 million reads per cell. Sequencing was carried out as paired-end reads of 75 bp length (PE75) with library indexes corresponding to cell barcodes.