GSM5237089: Stat1_4 (BRBseq); Mus musculus; RNA-Seq
Direct-zol RNA kit (Zymo Research, Cat. #R2052) was used to extract RNA according to the manufacturer's instruction. 3'end bulk mRNA cDNA library preparation and sequencing was performed following the BRB-seq strategy as previously described (Alpern et al., 2019). In brief, 20 ng of total RNA isolated from TF overexpressions from each sample was reverse transcribed using SuperScriptTM II Reverse Transcriptase (Lifetech, Cat. Cat. #18064014) with individual barcoded oligo-dT primers, featuring a 12-nt-long sample barcode (IDT). Double-stranded cDNA was generated by second strand synthesis via the nick translation method. To that end, a mix containing 2 μl of RNAse H (NEB, Cat. #M0297S), 1 μl of E. coli DNA ligase (NEB, Cat. #M0205 L), 5 μl of E. coli DNA Polymerase (NEB, Cat. #M0209 L), 1 μl of 10mM dNTP  (Thermo Fisher Scientific, Cat. #0181), 10 μl of 5x Second Strand Buffer (100 mM Tris, pH 6.9, [AppliChem, Cat. #A3452], 25 mM MgCl2 [Sigma, Cat. #M2670], 450 mM KCl [AppliChem, Cat. #A293], 0.8 mM β-NAD [Sigma, Cat. N1511], 60 mM (NH4)2SO4 [Fisher Scientific, Cat. #AC20587]), and 11 μl of water was added to 20 μl of ExoI-treated first-strand reaction on ice. The reaction was incubated at 16°C for 2.5 h. Full-length double-stranded cDNA was purified with 30 μl (0.6x) of AMPure XP magnetic beads (Beckman Coulter, Cat. #A63881) and eluted in 20 μl of water. cDNA concentration was measured using Qubit, and cDNA quality was assessed using a Fragment Analyzer (Agilent). cDNA was tagmented with in-house Tn5 (Picelli et al., 2014), and libraries were purified using AMPure XP magnetic beads (0.6X). The resulting libraries were profiled with a High Sensitivity NGS Fragment Analysis Kit (Advanced Analytical, Cat. #DNF-474) and measured with the Qubit dsDNA HS Assay Kit (Invitrogen, Cat. #Q32851) Sequenced using the Illumina NextSeq 500 platform utilizing a BRB-seq custom primer and the High Output v2 kit (75 cycles) (Illumina, Cat. #FC-404-2005). The sequencing configuration is as follows: Read1 21cycles / index i7 8cycles / Read2 63c.