title: GSM6246299: KPC WT ECM Q-3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA were extracted using All Prep DNA/RNA Mini Kit (Qiagen). RNA purity was assessed by an Agilent 2100 Bioanalyzer. 500 ng total RNA was used for the construction of sequencing libraries. 500 ng total RNA was enriched for poly-A tailed RNA transcripts by double incubation with Oligo d(T) Magnetic Beads (NEB, S1419S) and fragmented for 9 min at 94ºC in 2X Superscript III first-strand buffer containing 10 mM DTT (Invitrogen, #P2325). Reverse-transcription (RT) reaction was performed at 25ºC for 10 min followed by 50ºC for 50 min. RT product was purified with RNAClean XP (Beckman Coulter, #A63987). Libraries were ligated with dual UDI (IDT) or single (Bioo Scientific), PCR-amplified for 11-13 cycles, size-selected using one-sided 0.8× AMPure cleanup beads, quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and sequenced on a HiSeq 4000. RNA-seq reads were aligned to the mouse genome (GRCm38/mm10) using STAR.