GSM6327016: mouse kidney, RC-Casp1KO, rep 4; Mus musculus; RNA-Seq
RNA was harvested using TRIZOL, followed by DNase  treatment, followed by cleanup with RNeasy column (Qiagen). 5 µg of total RNA was used for the construction of sequencing libraries. mRNA was purified from total RNA using poly-T magnetic beads. The mRNA was fragmented, and first strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was performed using DNA Polymerase I and RNase H. Double-stranded cDNA was purified using AMPure XP beads (Beckman Coulter). Remaining overhangs of the purified double-stranded cDNA were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. Library fragments were purified with AMPure XP system (Beckman Coulter) to select cDNA fragments ~150-200 bp. The final library was generated by PCR amplification and purification of PCR products by AMPure XP beads.