title: GSM8861537: No ASC control Replicate 2; Mus musculus; RNA-Seq
library_name: GSM8861537
library_construction_protocol: RNA was preserved in RNALater (Invitrogen) at 4°C overnight, or for up to one week, according to the manufacturer's protocol, to ensure RNA quality was not compromised. The lung tissues were then removed from RNAlater™ and stored at -80°C. Total RNA was isolated from 15 – 18 mg of these lung tissues. The lung tissues were then added to 600 µL of RLT buffer, supplemented with 10uL of β-Mercaptoethanol (MP Biomedicals, Santa Ana, CA), and homogenized using a PRO200 homogenizer (MidSci, ValleyPark, MO) in three pulses of 10-15 s each at mid-power (setting 3). For cell pellets collected from in vitro experiments, 350 µL of RLT buffer was added to each sample and homogenized by passing the cells through a 27G tuberculin syringe three times. Lysates collected fromin vivo experiments were processed using the Qiagen RNeasy kit (Qiagen, Germantown, MD) and eluted in 50 μL of Ultrapure DNAse/RNase-free distilled water (Invitrogen). Poly(A) RNA sequencing library was prepared following Illumina's TruSeq-stranded-mRNA sample preparation protocol. RNA integrity was checked with Agilent Technologies 2100 Bioanalyzer. Poly(A) tail-containing mRNAs were purified using oligo-(dT) magnetic beads with two rounds of purification. After purification, poly(A) RNA was fragmented using divalent cation buffer in elevated temperature. The DNA library construction is shown in the following workflow. Quality control analysis and quantification of the sequencing library were performed using Agilent Technologies 2100 Bioanalyzer High Sensitivity DNA Chip.