title: GSM1635137: Mast cells stimulated; Mus musculus; RNA-Seq
library_construction_protocol: Mast cell total RNA was isolated using Trizol Barcoded mRNA-seq cDNA libraries were prepared from 5ug of total RNA using a modified version of the Illumina mRNA-seq protocol. With the aid of Seramag Oligo(dT) magnetic beads (Thermo Scientific) mRNA was isolated and subsequently fragmented using divalent cations and heat resulting in fragments ranging from 160-220bp. Fragmented mRNA was converted to cDNA using random primers and SuperScriptII (Invitrogen) followed by second strand synthesis using DNA polymerase I and RNaseH. cDNA was end repaired using T4 DNA polymerase, Klenow DNA polymerase and 5’ phosphorylated using T4 polynucleotide kinase. Blunt ended cDNA fragments were 3’ adenylated using Klenow fragment (3’ to 5’ exo minus). 3’ single T-overhang Illumina multiplex specific adapters were ligated using a 10:1 molar ratio of adapter to cDNA insert using T4 DNA ligase. cDNA libraries were purified and size selected at 200-220bp using the E-Gel 2% SizeSelect gel (Invitrogen). Enrichment, adding of Illumina six base index and flow cell specific sequences were done by PCR using Phusion DNA polymerase (Finnzymes). Clean-up was done using 1,8x volume of AgencourtAMPure XP magnetic beads. All quality controls were done using Invitrogen’s Qubit HS assay and fragment size was determined using Agilent’s 2100 Bioanalyzer HS DNA assay. Barcoded RNA-Seq libraries were clustered on the cBot using Truseq SR cluster kit v2.5 using 7pM and 50bps were sequenced on the Illumina HiSeq2000 using Truseq SBS kit-HS 50bp. The raw output data of the HiSeq was preprocessed according to the Illumina standard protocol
attributes.tag: GEO Accession
attributes.value: GSM1635137