GSM5834190: Sample 12; Mus musculus; RNA-Seq
Cells were washed once with PBS before harvesting. For the RNA samples, cells were collected with TRIzol Reagent (Invitrogen, 15596018) or RLT buffer (Qiagen, 74004) + B-mercaptoethanol. Lysates were homogenized and stored at -20°C. Samples were thawed and 0.2 mL chloroform per 1 mL of TRIzol was added and incubated for 2–3 minutes. Samples were centrifuged for 15 minutes at 12000 g at 4°C and the aqueous phase was transferred to a new tube. From this aqueous phase the RNA was isolated using RNeasy Micro Kit (Qiagen, 74004) following the manufacturer's instructions. RNA was eluted in 14uL RNase-free water and RNA yield was determined by nanodrop. RNA quality was determined using the Bioanalyzer (Agilent) and the RNA 6000 Nano Kit (Agilent, 5067-1511). Technical triplicates were pooled, resulting in a total of three biological replicates per conditions. All samples were diluted to a concentration of 40 ng/uL and shipped in dry ice to BGI tech solutions (Hongkong). RNA preparation, library construction and sequencing on BGISEQ-500 was performed at Beijing Genomics Institute. At BGI tech solutions, DNBseq eukaryotic transcriptome re-sequencing (strand specific) was done applying at least 20M clean reads per sample. Filtered sequencing reads were collected (6-9 GB data per sample).