title: GSM3891027: WT_untreated_RepB; Mus musculus; RNA-Seq
library_construction_protocol: Cell pellets were gently lysed in 100 μL buffer RLN (per Qiagen RY25 protocol: 50 mM Tris-Cl (pH 8.0), 140 mM NaCl, 1.5 mM MgCl2, 0.5% (v/v) Nonidet P-40 Substitute (USB)) per 8 × 105 cells for 10 min on ice, and then centrifuged briefly at 16k rcf to remove nuclei and incompletely lysed cells. Total RNA from cytosolic lysates was extracted with 1 mL TRI reagent (Sigma) per 200 μl lysate with isopropanol precipitation per manufacturer's instructions. Each biological replicate for library construction was generated independently. One μg of total purified RNA from cytoplasmic fractions was enriched for poly-A-containing transcripts using Dynabeads mRNA DIRECT Purification kit (Life Technologies). mRNA was fragmented thermo-chemically in RT buffer (Agilent) in the presence of custom barcoded oligo-dT primer/adapters (IDT) at 94 C for exactly 4.5 min and immediately eluted from beads. Primers were allowed to anneal to transcripts at room temperature and cDNA was generated using AffinityScript Reverse Transcriptase (Agilent). Samples were normalized and pooled by relative levels of mActB transcripts. RNA from cDNA hybrids was degraded with 1M NaOH at 70 °C for 12 min and neutralized with 0.5 M acetic acid. 3' barcodes were removed by Agencourt AMPure XP beads (Beckman Coulter). Following ligation of a pre-adenylated 5' adapter (IDT) using Thermostable T4 ligase (NEB), and barcode cleanup, fragments were amplified using 12 cycles of enrichment with Fusion High-Fidelity Master Mix (NEB) with primers compatible with Illumina sequencing. Library quantity and quality was confirmed using High Sensitivity D1000 ScreenTape on a 2200 TapeStation (Agilent).