GSM3272213: Post_9_sans_F9; Homo sapiens; RNA-Seq
Cells were harvested after a 4 h incubation, and RNAs were collected in 1 ml of Trizol and stored at -80°C until RNA extraction for sequencing. Total RNA was isolated using Trizol method (Life Technologies SAS) combined with Rneasy mini kit procedure (Qiagen). RNA quality was assessed using the Agilent 6000 Pico Kit as instructed by the manufacturer. All RNA samples yield maximum quality with the RNA integrity number (RIN) above 9. Smart-Seq2 libraries were prepared by the Broad Technology Labs and sequenced by the Broad Genomics Platform according to the SmartSeq2 protocol (Picelli et al, 2013, 2014) with some modifications (Trombetta et al, 2014). Briefly, total RNA was purified using RNA-SPRI beads, polyA mRNA was converted to cDNA and amplified cDNA was subject to transposon-based fragmentation that used dual-indexing to barcode each fragment of each converted transcript with a combination of barcodes specific to each sample. Sequencing was carried out as paired-end 2x25bp with an additional 8 cycles for each index.