GSM7432645: PRO-seq.mESC.Mut.1vsWT_WT_rep1; Mus musculus; OTHER
N.A PRO-Seq library construction and data analysis was performed by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA, as follow:   Aliquots of frozen (-80°C) permeabilized cells were thawed on ice and pipetted gently to fully resuspend. Aliquots were removed and permeabilized cells were counted using a Luna II, Logos Biosystems instrument. For each sample, 1 million permeabilized cells were used for nuclear run-on, with 50,000 permeabilized Drosophila S2 cells added to each sample for normalization. Nuclear run on assays and library preparation were performed essentially as described in Reimer et al. [K. A. Reimer, C. A. Mimoso, K. Adelman, K. M. Neugebauer, Molecular Cell (2021)]  with modifications noted: 2X nuclear run-on buffer consisted of (10 mM Tris (pH 8), 10 mM MgCl2, 1 mM DTT, 300mM KCl, 20uM/ea biotin-11-NTPs (Perkin Elmer), 0.8U/uL SuperaseIN (Thermo), 1% sarkosyl).  Run-on reactions were performed at 37°C.  Random hexamer extensions (UMIs) were added to the 3' end of the 5' adapter and 5' end of the 3' adapter.  Adenylated 3' adapter was prepared using the 5' DNA adenylation kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturers' instructions with 15% PEG-8000 final) and incubated at 16°C overnight.  180uL of betaine buffer (1.42g of betaine brought to 10mL) was mixed with ligations and incubated 5 min at 65°C and 2 min on ice prior to addition of streptavidin beads.  After T4 polynucleotide kinase (NEB) treatment, beads were washed once each with high salt, low salt, and 0.25X T4 RNA ligase buffer (NEB) and resuspended in 5' adapter mix (10 pmol 5' adapter, 30 pmol blocking oligo, water).  5' adapter ligation was per Reimer but with 15% PEG-8000 final. Eluted cDNA was amplified 5-cycles (NEBNext Ultra II Q5 master mix (NEB) with Illumina TruSeq PCR primers RP-1 and RPI-X) following the manufacturer's suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries. Pooled libraries were sequenced using the Illumina NovaSeq platform.