GSM7884586
Cell pellets were resuspended in 1ml TRIzol, then stored at -80°C. Samples were heated at 65°C for 10 min. 0.2 ml chloroform was mixed into samples and incubated for 5 min. Samples pelleted at 17,000xg for 15 min at 4°C. Aqueous layer transferred to fresh tube. 450 µl isopropanol was added and stored at -80°C overnight. Thawed samples were pelleted at 4°C for 30 min, then supernatant was removed. Pellets were washed in 1 ml 70% ethanol, dried, and resuspended in 100 µl RNase-free H2O. Samples were cleaned up with RNeasy Mini Kit (QIAGEN). Samples were DNAse treated with Invitrogen DNAse (RNAse free). Library preparation was performed using Illumina's Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit and 10bp IDT for Illumina indices. Sequencing was done on a NextSeq2000 giving 2x51bp reads. Demultiplexing, quality control, and adapter trimming was performed with bcl-convert (v3.9.3)