GSM3770727: RNA_B6C3F1_Vh_1032; Mus musculus; RNA-Seq
RNA-extraction: 25-50 mg of frozen liver material was used for total RNA-extraction. Samples were homogenized in TRIzol reagent (ThermoFisher, cat.No. 15596026) and total RNA was isolated by phase separation using chloroform-isoamyl alcohol. RNA precipitation with isopropyl alcohol was performed and precipitated RNA was resuspended in EB buffer (Qiagen). Samples were NanoDrop quantified and the RNA Integrity Number (RIN) measured using an Agilent RNA 6000 Nano Kit (Agilent, cat.No. 5067-1511) on a BioAnalyzer instrument. Samples with RIN above 8 were used for sequencing. DNase-Hypersensitivity: 100-150 mg of frozen liver tissue was homogenized in ice-cold Nuclear Homogenization Buffer (NEHB) using a 7 ml dounce tissue homogenizer. Nuclei were isolated by ultracentrifugation, overlying the homogenized tissue on top of 5 ml NEHB. Isolated nuclei were rinsed three times in 1 ml ice-cold Buffer A. The pelleted nuclei were resuspended in Digestion buffer. DNase I digestion was carried out on 107 nuclei per mouse at 37 °C for 3 min using a final concentration of 50 U/ml of RQ1 RNase-free DNase I  (Promega, cat.no M6101). DNase digestion was stopped and DNA purification was performed. DNA fractionation was performed on the recovered extraction solution using a sucrose step gradient (40%, 35%, 30%, 25%, 20%, 17.5%, 15%, 12.5%, and 10% solutions in 1x Centrifugation buffer). After ultracentrifugation, the first 3 ml fraction was recovered and purified over a MinElute Purification Column (Qiagen). RNA-seq: Illumina libraries were prepared with the TruSeq Stranded Total RNA Sample Preparation kit with Ribo-Zero Gold. We generated paired-end 76 bp reads on a HiSeq 2500 using v4 reagents (all Illumina). DNase-seq: Next generation sequencing libraries were prepared with NuGEN Ovation Ultralow System V2 with A-tailing (TECAN, Leek, The Netherlands). Size selection of the 40 – 250 bp fraction on a BluePippin BDF3010 3% DF Marker Q2 cassette or PippinHT BEF2010 2% DF Marker M1 cassette (both Sage Science, Beverly, MA, USA), and paired-end 76 bp sequencing was performed on a HiSeq 2500 using v4 reagents (all Illumina).
GEO Accession
GSM3770727