title: GSM4478937: KKO Rep1 PROSeq; Myotis velifer; RNA-Seq
library_construction_protocol: PRO-seq libraries were prepared as previously described (Judd, et al. 2020; Mahat, et al. 2016; Kwak, et al. 2013). In brief, cells were grown to approximately 80% confluency and were chilled on ice, washed twice with ice cold PBS, incubated in PBS with 1 mM EDTA for 5 minutes, and harvested by scraping. Cells were permeabilized by incubating on ice for 5 minutes in permeabilization buffer (10 mM Tris-Cl pH 7.5, 10 mM KCl, 250 mM Sucrose, 5 mM MgCl2, 1 mM EGTA, 0.5 mM DTT, 0.05% Tween-20, 4 U/mL SUPERase-In [Thermo Fisher], 1X EDTA Free Pierce Protease Inhibitors [Thermo Fisher], and 0.1% NP-40). Permeabilization was verified using Trypan blue staining followed by visual inspection. Cells were washed once with permeabilization buffer and flash frozen in LN2 in storage buffer (50 mM Tris-Cl pH 8.0, 40% v/v Glycerol, 5 mM MgCl2 0.1 mM EDTA, and 0.5 mM DTT). Nuclear Run-On was performed for 5 minutes at 37˚C by mixing equal volumes of permeabilized cells in storage buffer with 2X Run-On buffer (10 mM Tris-Cl pH 8.0, 5 mM MgCl2, 1 mM DTT, 300 mM KCl, 40 µM ATP, 40 µM GTP, 40 µM Biotin-11-CTP [Perkin Elmer], 40 µM Biotin-11-UTP [Perkin Elmer], 2 U/mL SUPERase-In [Thermo Fisher], and 1% Sarkosyl). RNA was isolated by TRIzol [Thermo Fisher] extraction and was fragmented by base hydrolysis in 0.2 N NaOH on ice for 5 min. Following neutralization with pH 6.8 Tris-Cl, RNA was passed through an RNase free Bio-Gel P-30 column [Bio-Rad]. The 3' and 5' adapters used were identical to those in (Mahat, et al. 2016) except 6 random nucleotides were added at the ligation junction as a unique molecular identifier (UMI) to facilitate computational removal of PCR duplicates. The 3' adapter was ligated to the total RNA pool (containing both nascent and non-nascent RNAs) for 1 h at 25 ˚C using 30 U T4 RNA ligase 1 [NEB] in 10% PEG8000. Nascent RNA was then captured using Dynabeads MyOne C1 Streptavidin beads [Thermo Fisher] and washed once with high salt wash buffer and once with low salt wash buffer as described in (Mahat, et al. 2016). RNA was 5' phosphorylated while attached to the beads using T4 PNK [NEB] and then 5' uncapped using RppH [NEB] while attached to the beads. RNA was then eluted from the beads using TRIzol, the 5' adapter was ligated using 10 U T4 RNA Ligase 1 for 1 h at 25 ˚C. RNA was again isolated using Dynabeads MyOne C1 Streptavidin beads, and reverse transcription was performed on the beads using Maxima H- RT [Thermo Fisher]. cDNA was eluted from the beads by heating, and final libraries were amplified for 11 PCR cycles using Q5 High Fidelity DNA Polymerase [NEB]. Libraries were sequenced on an Illumina NextSeq 500 platform using paired end 37 by 37-bp chemistry. library strategy: PRO-Seq
attributes.tag: GEO Accession
attributes.value: GSM4478937