GSM7727173: maB_mRNA_r3_0h; Mus musculus; RNA-Seq
RNA-Seq Cells (0.5M) were harvested and lysed in 100 µl of the lysis solution (RNAqueous™-Micro Total RNA Isolation Kit from Invitrogen). RNA was isolated and DNase treated according to manufacturer's protocol. High RNA quality was confirmed using the Agilent TapeStation system before library preparation. mRNA polyA- purification, reverse transcription and library preparation were performed using NEBNext Poly(A) mRNA Magnetic Isolation Module. For spike-in control ERCC RNA Spike-In Mix (Invitrogen) was added before RNA isolation.   ChIP-Seq Cells were fixed with 1% formaldehyde (Sigma) for 10 min at 37°C, treated with 1/20 volume of 2.5 M Glycine, washed with PBS, snap-frozen on dry ice and stored at -80°C until further processing. After thawing cells were resuspended in RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100) supplemented with Complete Mini EDTA free proteinase inhibitor (Roche) and sonicated using ultrasonicator (Covaris) in 1 ml AFA tubes (Covaris): duration (s): 1200, peak power: 75, duty % factor: 15, cycles/burst:1000. Sonicated chromatin was pre-cleared with Dynabeads and incubated overnight at 4˚C with rotation with following antibodies: RAD21: ab992 (Abcam); MED26: 13641S (Cell Signaling Technology); TATA binding protein TBP: ab28175 (Abcam); BRD4: ab128874 (Abcam); RNA Pol II: ab26721 (Abcam). Next day samples were washed (10 min at 4˚C with rotation): 2x with RIPA buffer, 2x with RIPA buffer + 0.3 M NaCl, 2x with 1 ml of LiCl buffer (0.25M LiCl, 0.5% Igepal CA-630, 0.5% NaDOC, store at 4˚C), 1x with 1 ml of TE + 0.2 % Triton X-100, 1x with 1 ml of TE. After washes beads were resuspended with 100 ul of TE, followed by addition of 3 µl of 10% SDS and 5 µl of 20 mg/ml Proteinase K and incubated at 65˚C for 4h. After decrosslinking DNA was purified on columns using ChIP DNA Clean & Concentrator kit (Zymo Research). In the next step DNA was quantified using Qubit system (Invitrogen).  mRNA half-lives estimation  Cells were treated with 5 µg/mL actinomycin D (Sigma) and harvested (0.5M cells) at timepoint 0 hours and up to 24 hours: at 1, 2, 4, 8, 12 and 24 hours timepoints7. After each timepoint the viability of cells was checked with FACS, or Nexcelom cell counter after staining with Acridine Orange/Propidium Iodide (AO/PI) (Nexcelom Bioscience). Some cell types did not survive longer actinomycin D treatment than 8 hours, so the last collected timepoint was determined based on cell type viability (details in Extended Data Fig.1a). Pellets were lysed in 100 µl of the lysis solution (RNAqueous™-Micro Total RNA Isolation Kit from Invitrogen), ERCC RNA Spike-In Mix (Invitrogen) was added, RNA isolated (RNAqueous™-Micro Total RNA Isolation Kit from Invitrogen), and RNA-Seq was performed using NEBNext Poly(A) mRNA Magnetic Isolation Module.   scRNA-Seq Cells were washed twice with 1x PBS containing 0.04% weight/volume BSA, resuspended in the same buffer and encapsulated into droplets using 10X Genomics system. RNA-Seq NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used for library construction. Single 50 cycles of sequencing data were acquired on NextSeq2000, NextSeq550, HiSeq3000 or NovaSeq6000 (Illumina).  ChIP-Seq Library was prepared using Ovation Ultralow System V2 (Tecan) according to manufacturer's protocol. Before sequencing high quality was confirmed using the Agilent TapeStation system. Single 50 cycles of sequencing data were acquired on NextSeq550, NovaSeq6000 (Illumina).  scRNA-Seq Libraries were prepared using Chromium Single Cell 3′ Reagent Kits (10X Genomics) according to the manufacturer's protocol. With double 10bp index cycles, 28 forward and 90 reverse cycles were run on NovaSeq6000 machine (Illumina).