GSM2052630: 2776-TL-0004; Mus musculus; RNA-Seq
After 48 hr of seeding on substrates, cells were lysed using QIAzol Lysis Reagent (Qiagen Sciences, Germantown, MD USA) following the manufacturer’s protocol. The cell lysate was immediately stored at -80°C and transported on dry ice to the Genomic Services Laboratory (HudsonAlpha, Huntsville, AL, USA) for RNA isolation and sequencing. Total RNA containing both mRNA as well as microRNA fractions was extracted from the cell lysate using the miRNeasy Kit with on-column DNase treatment (Qiagen Inc., Valencia, CA, USA) according to the manufacturer's protocol. The final elution was performed in 30 µl of RNase-free sterile distilled water. The concentration and integrity of the extracted total RNA were estimated using the Qubit® 2.0 Fluorometer (Invitrogen)and Agilent 2100 Bioanalyzer (Applied Biosystems, Carlsbad, CA, USA), respectively. RNA samples with RNA integrity numbers (RIN) of at least 7.0 were used for further processing. Five hundred nanograms of total RNA for samples with RIN values of greater than 7.0 was required for downstream RNA-seq applications. One microliter of a 1:100 dilution of ERCC spike-in consisting of either Mix 1 or Mix 2 (Invitrogen) was added to each sample. Polyadenylated RNAs were isolated using NEBNext Magnetic Oligo d(T)25 Beads. The NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs Inc., Ipswich, MA, USA) was used to prepare individually bar-coded next-generation sequencing expression libraries. Library quality was assessed using the Qubit 2.0 Fluorometer, and the library concentration was estimated by utilizing a DNA 1000 Chip on an Agilent 2100 Bioanalyzer. Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Each library was diluted to a final concentration of 12.5 nM and pooled in an equimolar ratio prior to clustering. Paired-end sequencing (25 million reads per sample, 50-bp) was performed on an Illumina HiSeq2500 sequencer (Illumina, Inc., San Diego, CA, USA)
GEO Accession
GSM2052630