GSM5032104: WT_1 ATRi L5 [SLX-17502.NEBNext04.H5J7KBBXY.s_5]; Mus musculus; RNA-Seq
RNA was extracted using a RNeasy kit with on column DNase digestion according to the manufacturer's protocol (Qiagen, 74106). RNA integrity was confirmed using Agilent Tapestation RNA screentape, showing 2 sharp peaks representing the 18S and 28S ribosomal subunits, with a 28S:18S ratio of approximately 2. Using 5 mg input RNA, mRNA was isolated from ribosomal RNA using the NEBNext Poly(A) mRNA magnetic isolation module (E7490) according to the manufacturer's instructions. Following two rounds of enrichment, successful isolation and sample integrity (RINe 2-3) were confirmed using high sensitivity Agilent Screen tape. Library preparation was performed using the NEBNext Ultra II directional library prep kit (E7760) according to the manufacturer's instructions for “purified RNA or clean RNA”. cDNA concentration was measured using the Qubit dsDNA assay kit (Q32854), and molar concentrations calculated using the average DNA fragment size following analyses on the Agilent Tapestation with high sensitivity DNA screentape (D1000). Equal molar concentrations were multiplexed at a final concentration of 15 nM and sequenced using Illumina Nextera Truseq single-ended reads. Due to limitations in the number of samples that could be combined in a single multiplex for sequencing, WT samples were independently multiplexed with Ccnc KO samples, and again with Cdk8 KO samples, in order to eliminate any sequencing-run bias.
GEO
GSM5032104