GSM2807926: E2-cyto; Homo sapiens; RNA-Seq
Unless mentioned otherwise, everything should be performed on ice or at 4°C and all solutions should be ice-cold. As adapted from Gagnon et al 2014, cells were washed with 20ml PBS and first scraped in 30 ml PBS then in 15ml for a second time. Cells were pelleted for 3 min at 1500 rpm, washed in PBS and aliquoted at approx. 107 cells per microtube. Cells were pelleted again for 3 min at 800g and resuspended in 380µl HLB hypotonic lysis buffer (10mM TrisHCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.3% NP40, 10% glycerol) supplemented with 25µg/ml alpha-amanitin as well as RNase-inhibitor and proteinase inhibitor. Cells were lysed for 10 minutes and centrifuged for 3 min at 1000g. The cytoplasmic fraction was recovered and stored at -20°C after adding 1ml of RPS RNA precipitation solution (10 mM Na2Ac, 95% EtOH). The nuclei pellet was then washed 3 times by adding 1 ml HLB and centrifugating for 2 min at 300g. Then nuclei-pellet was lysed by resuspending in 380 µl of MWS Modified Wuarin Schibler buffer (10 mM Tris-HCl pH 7, 4 mM EDTA, 0.3 M NaCl, 1 M urea, 1% NP-40) supplemented with 25µg/ml alpha-amanitin as well as RNase-inhibitor and proteinase inhibitor, vortexing for 30 sec on medium-speed, incubating on ice for 5 min , vortexing again for 30 sec and incubating once more for 10 min. Chromatin was pelleted for 3 min at 1000 g and nucleoplasmic fraction was recovered and stored in RPS at -20°C. Chromatin pellet was washed 3 times by adding 1 ml MWS, vortexing briefly on medium-speed and centrifugating for 2 min at 500g. Finally, 700µl of Qiazol was added to the pellet and stored at -20°C. The next day, cytoplasmic and nucleoplasmic fraction were centrifuged for 15 min at 14 000g, washed in 1 ml 70% EtOH and centrifuged again for 5 min at 14 000g. After air-drying the pellets, they were resuspended in Trizol. After unfreezing on ice, 10µl of 0.5M EDTA were added to the chromatin fraction. All fractions were incubated at 65°C for approx. 5 min and cooled at room temperature before proceeding to DNase treatment and RNA extraction using the QIAGEN miRNeasy Mini Kit according to the manufacturer's instructions. Total RNA-seq libraries were prepared with TruSeq Stranded Total RNA Sample Prep Kit (RS-122-2201, Illumina)
GEO Accession
GSM2807926