GSM8136797: MPRA TSR TF motif mutation DNA library; Homo sapiens; OTHER
For all csRNA, RNA-seq, and TSS-MPRA experiments, to harvest RNA, plates were washed three times using ice cold PBS and lysed using 1 ml TRIzol™ Reagent. Lysate was scraped and RNA extracted as described by the manufacturer with addition of 1 μl 15 mg/ml GlycoBlue™ Coprecipitant (Thermo Fisher Scientific) during the Isopropanol precipitation. For ChIP-seq, cells were fixed for 10 minutes with 1% formaldehyde/PBS, and the extracted chromatin was sheared to an average DNA size of 300–500 bp. csRNA-seq was performed as described in (Duttke et al. 2019). Small RNAs of ~20-60 nt were size selected from 0.4-2 µg of total RNA by denaturing gel electrophoresis. A 10% input sample was taken aside and the remainder enriched for 5'-capped RNAs. Monophosphorylated RNAs were selectively degraded by 1 hour incubation with Terminator 5´-Phosphate-Dependent Exonuclease (Lucigen). Subsequently, RNAs were 5'dephosporylated through 90 minutes incubation in total with thermostable QuickCIP (NEB) in which the samples were briefly heated to 75°C and quickly chilled on ice at the 60 minutes mark. small RNA input (csRNAinput-seq) and csRNA-seq libraries were prepared as described in (Hetzel et al. 2016) using RppH (NEB) and the NEBNext Small RNA Library Prep kit, amplified for 14 cycles and sequenced SE75 on the Illumina NextSeq 500. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq kit Stranded Total RNA Library kit (Illumina) and sequenced PE100 on Illumina NovaSeq 6000. For ChIP-seq, one percent of the lysate was kept as ChIP input, and dnNFR1-HA was immunoprecipiated with 2 μg anti-HA antibody (Abcam ab9110) and 20 μl of Dynabeads Protein A. Libraries were prepared directly on the antibody/chromatin-bound beads using NEBNext Ultra II reagents according to the manufacturer's protocol. Libraries were sequenced single-end for 75 cycles (SE75) to a depth of 20-25 million reads on an Illumina NextSeq 500 instrument. For TSS-MPRA, starting with 10-15 µg RNA (⅓ of the total), non-capped RNAs were dephosphorylated with Quick CIP (NEB) and DNA digested. Samples were mixed well and incubated at 37°C for 90 minutes. For more complete dephosphorylation of uncapped transcripts, RNA was denatured a second time at 75°C for 30 seconds in a pre-warmed water bath and then quickly chilled on ice for 2 minutes before incubating the sample at 37°C for another 30 minutes. As Quick CIP is thermolabile, the brief heat treatment leaves the enzyme active. Following Cap-enrichment, RNA was purified and then vortexed hard and subsequently spun for 10 minutes at 12k g at RT. Following phase separation, the upper layer was taken, and RNA precipitated. RNA pellets were then resuspended and 5' caps removed. Following decapping, 5' adapters were ligated by T4RNA Ligase 1 and RNA extracted. RNA pellets were then resuspended and reverse transcribed. Samples were amplified and sequenced paired-end on a NextSeq 500 (additional details available in the manuscript)