GSM4417418: CB2_UT_B; Homo sapiens; RNA-Seq
Cells were washed in PBS on ice and RNA was extracted with Trizol (Invitrogen). RNA purification was done using the Direct-zol TM RNA MiniPrep kit (catalog # R2050, Zymo Research). After QC procedures inclusive of agarose gel electrophoresis and ensuring RIN values of all samples were 9.8 or 10, mRNA was enriched using oligo(dT) beads. The mRNA was then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The final cDNA library was ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then are diluted to 1 ng/ul before checking insert size on an Agilent 2100 and further quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM). Libraries were fed into Illumina machines after pooling according to activity and expected data volume if library quality is up to standard.
GEO Accession
GSM4417418