GSM5124316: PC9DR wth Dacomitinib 2; Homo sapiens; RNA-Seq
QIAGEN miRNeasy Mini Kit Libraries for RNA-seq were prepared using a modified version of TranSeq, as described (Jaitin et al., 2014). Briefly, RNA was reversed transcribed with MARS-seq barcoded RT primers in a 10 µl volume with the Affinity Script kit (Agilent). Reverse transcription was analyzed by qRT-PCR and samples with a similar CT were pooled (up to eight samples per pool). Each pool was treated with Exonuclease I (NEB) for 30 min at 37°C and cleaned by 1.2× volumes of SPRI beads (Beckman Coulter). Next, the cDNA was converted to double-stranded DNA with a second strand synthesis kit (NEB) in a 20 ml reaction, incubating for 2 h at 16 °C. The product was purified with 1.4× volumes of SPRI beads, eluted in 8 µl and in vitro transcribed (with the beads) at 37 °C overnight for linear amplification using the T7 High Yield RNA polymerase IVT kit (NEB). Following IVT, the DNA template was removed with Turbo DNase I (Ambion) 15 min at 37 °C and the amplified RNA (aRNA) purified with 1.2 volumes of SPRI beads. The aRNA was fragmented by incubating min at 70 °C in Zn 621 2+ RNA fragmentation reagents (Ambion) and purified with 2× volumes of SPRI beads. The aRNA was ligated to the MARS-seq ligation adapter with T4 RNA Ligase I (NEB).  The reaction was incubated at 22 °C for 2 h. After 1.5× SPRI cleanup, the ligated product was reverse transcribed using Affinity Script RT enzyme (Agilent) and a primer complementary to the ligated adapter. The reaction was  incubated for 2 min at 42°C, 45 min at 50°C, and 5 min at 85°C. The cDNA was purified with 1.5× volumes of SPRI beads. The library was completed and amplified through a nested PCR reaction with 0.5 mM of P5_Rd1 and P7_Rd2 primers and PCR ready mix  (Kappa Biosystems). The amplified pooled library was purified with 0.7× volumes of SPRI beads to remove primer leftovers. Library concentration was measured with a Qubit fluorometer (Life Technologies) and mean molecule size was determined with a TapeStation instrument. RNA-seq libraries were sequenced using the Illumina NextSeq® 500 High Output v2 Kit (75 cycles).