title: GSM8120925: HEK293CDK7as_STARTseq_DMSO_30min_Rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Start-seq: Start-seq was performed as previously described (Nechaev et al. 2010) with minor changes. 3.5x10^7 CDK7as cells were treated for 30 min with 7.5 μM of 1-NM-PP1 or DMSO (solvent control). For spike-in normalization, 2.5 x10^7 Drosophila S2 cells were added to each sample. After the treatment, nuclei isolation was performed as described in the protocol for cellular fractionation (Conrad et al. 2017). All buffers were supplemented with 7.5 μM 1-NM-PP1 or respective amounts of DMSO, protease (Sigma-Aldrich, P8340), and phosphatase (Roche, 4906837001) inhibitors. RNA was extracted from the isolated nuclei using 5 ml of TRIzol reagent (Invitrogen, 15596026), precipitated with 100% ethanol and GlycoBlue coprecipitant (Invitrogen, AM9515) at -20°C for 30 minutes, then washed twice with 70% ethanol. The RNA pellets were air-dried and dissolved in 7 M urea. To size select 16-65 nt RNA, samples were resolved on 9% polyacrylamide gel containing 7 M urea. RNA of corresponding sizes was extracted from the gel and precipitated with 100% ethanol and GlycoBlue coprecipitant. After short RNA isolation, uncapped pRNAs were degraded using Terminator 5′ phosphate-dependent exonuclease (Biozym, 162370) at 30°C for 60 min. After the reaction, short RNA was cleaned up using 1.8x of AMPure RNAClean XP (Beckman Coulter, A63987) beads and 270 µl of isopropanol. Next uncapped pppRNAs and pRNAs were dephosphorylated using rSAP (NEB, M0371) at 37°C for 30 min. RNA was cleaned up using the AMPure RNAClean XP beads, and RNA was uncapped with Cap-Clip (Biozym, C-CC15011H) at 37°C for 60 min. RNA was cleaned up using the AMPure RNAClean XP beads. Start-seq: 44 ng of RNA was used for library preparation with TruSeq Small RNA Library Prep Kit (RS-200-0048) according to the manufacturer's instructions. Fragment sizes were analyzed with the Fragment Analyzer (HS NGS Fragment Kit).