GSM3168967: JTG00333_wt; Mus musculus; RNA-Seq
Pneumocytes were collected directly in Tris Reagent and stored at -80ºC. RNA was then isolated using PicoPureTM RNA isolation kit. RNA sequencing libraries were prepared following the SMARTseq2 protocol (Picelli et al., 2013) with some modifications. Briefly, RNA quality (the RIN value) and quantity was estimated from the Agilent RNA 6000 Nano assay and 1.8ul of RNA was used for cDNA synthesis. Reverse transcription was performed using SuperScrpit II (Invitrogen) in the presence of oligo-dT30VN (1µM; 5′-AAGCAGTGGTATCAACGCAGAGTACT30VN-3′), template-switching oligonucleotides (1µM) and betaine (1M). The cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kappa Biosystems), 100 nM ISPCR primer (5′-AAGCAGTGGTATCAACGCAGAGT-3′) and 8 or 20 cycles of amplification, in function of the starting material quantities. Following purification with Agencourt Ampure XP beads (1:1 ratio; Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitvity DNA Kit (Agilent). 200 ng of the amplified cDNA was fragmented for 10 min at 55 ºC using Nextera® XT (Illumina) and amplified for 12 cycles with indexed Nextera® PCR primers. The Nextera® libraries were sequenced on HiSeq2000 (Illumina, Inc) in paired-end mode with a read length of 2x76bp using TruSeq SBS Kit v4 and TruSeq Dual Index of 8bp+8bp. We generated in a mean 20 million paired-end reads for each sample in a fraction of a sequencing v4 flow cell lane, following the manufacturer's protocol. Image analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.18.66.3) and followed by generation of FASTQ sequence files by CASAVA.Products were purified twice with Agencourt Ampure XP beads (0.8:1 ratio) and quantified again on a Bioanalyzer using a High Sensitvity DNA Kit.
GEO Accession
GSM3168967