title: GSM4367408: A549_rep2_30min; Drosophila melanogaster; Homo sapiens; RNA-Seq
library_construction_protocol: At each 5 minute time point, one plate of cells was removed from the incubator, washed once in ice cold 1x PBS, then 1mL of Cytoplasmic Lysis Buffer + S2 cells (CL+S2 buffer, 25mM Tris pH 7.9, 150mM NaCl, 0.1mM EDTA, 0.1% TritonX, 1mM DTT, 1x protease inhibitor cocktail, 1.3x10^6 S2 cells/mL) was added directly to the plate. Cells were collected by scraping and kept on ice until the end of the time course. After all samples were collected and incubated in CL+S2 buffer on ice for at least 10 min, the lysates were centrifugated at 3000 RPM for 5 min at 4C. The supernatant was removed and the pellet was washed in 1 mL CL buffer (without S2 cells) and re-centrifugated at the same speed. The pellet was then resuspended in 100μL Glycerol Resuspension Buffer (GR buffer, 20mM Tris pH 7.9, 75mM NaCl, 0.5mM EDTA, 50% glycerol, 0.85mM DTT), to which 1.1mL Nuclear Lysis Buffer (NL buffer, 20mM HEPES pH 7.6, 300mM NaCl, 7.5mM MgCl2, 1% NP-40, 1mM DTT, 1M Urea) was subsequently added. The resulting lysate was incubated on ice (with regular vortexing) for 15 min, then centrifugated at 16,000G for 10 min at 4C. The resulting chromatin pellet was resuspended in TRIzol (Invitrogen, 15596018) and RNA was extracted according to the manufacturer's protocol. To ensure complete depletion of mRNA, the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, E7490L) was used to capture and remove polyA mRNA. The manufacturer's protocol was followed until the mRNA was bound to the beads. Following mRNA binding, the supernatant was removed from the beads and the nascent RNA was ethanol precipitated and resuspended in ddH2O. Nascent RNA was quantified on a Denovix QFX fluorometer using the Qubit RNA HS assay according to the manufacturer's protocol. The quality of the RNA was assessed using an Aligent 2100 Expert Bioanalyzer instrument. Sequencing libraries were generated using the KAPA Stranded RNA-seq Kit with RiboErase (HMR) (Roche 07962282001) according to the manufacturers protocol. Final libraries were also subjected to bioanalyzer runs to ensure proper fragment length distribution. All 9 libraries were barcoded and sequenced together on 2 lanes of an Illumina HiSeq (paired-end 150 bp reads).
attributes.tag: GEO Accession
attributes.value: GSM4367408