title: GSM3672214: RNA-Seq_C59D2, rep2; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted with the QIAGEN RNeasy Mini Kit according to manufacturer's instructions, lysing cells directly into 6-well plates with buffer RTL plus. 5 µg of the eluted RNA were treated with DNase I in a 25-µl reaction at 37 °C for 30' (Invitrogen DNA-free™ DNA Removal Kit). DNA-free RNA was quantified by nanodrop and quality checked by Bioanalyzer and 2.5 µg were subjected to ribosomal RNA depletion following Illumina Ribo-zero rRNA Removal Kit's instructions. Precipitated RNA was resuspended to 17 µl End Repair Mix (ERP) from the TruSeq RNA Sample Preparation v2 Kit (Illumina RS-122-2001) and stored overnight at -80 °C until library preparation. RNA fragmentation, first and second strand cDNA synthesis were performed according to the TruSeq RNA Sample Preparation v2 Kit but using Superscript III for reverse transcription instead of Superscript II (50 °C for 50' incubation time). cDNA was purified with AMPure XP beads diluted 1:2 with 20% PEG, 1.25M NaCl, and eluted in 38.5 μl 10 mM Tris-HCl pH 8.0, 36.5 μl of which were transferred to a new tube and subjected to the ChIP-Seq Solexa rapid library protocol (see linked manuscript).
attributes.tag: GEO Accession
attributes.value: GSM3672214