GSM6529259: HAEC, Altered Rapamycin, rep1; Homo sapiens; RNA-Seq
Following unimodal (flow) or bimodal (flow and chemical treatment) stimulation, HAEC were washed with PBS and 0.05% Trypsin-EDTA was injected. Cells were incubated for 5 min and collected in a microcentrifuge tube with complete medium. Cells were centrifugated (300g for 5 minutes) and resuspended for analysis. After whole-transcriptions-amplification, samples libraries were prepared for sequencing using the Nextera XT DNA tagmentation/amplification. Libraries were purified with SPRI ratios of 0.6x and 0.8x applied to the amplified and tagmented product, respectively. Final libraries had an average fragment size distribution between 400 and 600 bp. Libraries were loaded onto a NextSeq 500 at a final loading concentration of 2.2 pM with a read structure as follows: (read 1) 20 cycles covering the 12-bp cell barcode and 8-bp unique molecular identifiers (UMIs); (read 2): 50 cycles on the mRNA strand; (index 1) 8 cycles covering sample-specific barcode sequences. Each sample was allotted 200 million reads for a target sequencing depth of 10-15k reads per cell.