Similar protocols

Protocol publication

[…] RNA was isolated from vLGN and dLGN at four different ages (P3, P8, P12 and P25) and was shipped to the Genomics Research Laboratory at Virginia Tech’s Biocomplexity Institute for RNAseq analysis. Quality of total RNA was checked on Agilent BioAnalyzer 2100 (Agilent Technologies, Santa Clara CA). Libraries were generated using Apollo 324 Robot (Wafergen, CA). 500 ng of total RNA (with RIN ≥9.0) was enriched for polyA RNA using PrepX PolyA mRNA Isolation Kit (cat # 400047, Wafergen, Fremont, CA) and was then onverted into a library of template molecules using the PrepX RNA-Seq for Illumina Library Kit (cat # 400046, Wafergen, Fremont, CA). Validation of the 280–300 bp libraries (160–180 bp insert) was completed using an Agilent 2100 Bioanalyzer and quantitated using Quant-iT dsDNA HS Kit (cat # Q33120, Invitrogen). Eight individually indexed cDNA libraries were pooled and sequenced on an Illumina HiSeq, resulting in a minimum of 40–50 million reads. Libraries were clustered onto a flow cell using Illumina’s TruSeq PE Cluster Kit v3-cBOT-HS (cat # PE-401–3001), and sequenced 2 × 100 PE using TruSeq SBS Kit v3-HS (200-cycles) (cat # FC-401–3001). Low-quality base calls, sequences with low-complexity tails, and adaptor sequences were removed using a combination of Btrim and EA-utils. Sequencing reads were then aligned to the mouse genome (Tophat2/Bowtie) and expression determined via HTSeq counting. DESeq2 has been used to determine fold change and statistical significance of changes between samples. […]

Pipeline specifications

Software tools Btrim, ea-utils, TopHat, Bowtie, HTSeq, DESeq2
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus