title: GSM2828284: si-PADI2 RNA-seq rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA from T47D-MTVL cells either transfected with sicontrol versus siPadi2 or expressing wild type (WTr) versus R1810Ar mutant form RNAP2 was extracted with RNeasy columnsfrom Qiagen followed by DNase  treaments.          For chromatin RNA, cells were lysed for 5 minutes in 4ml of ice-cold HLB+N [10mM Tris-HCl pH 7.5, 10mM NaCl, 2.5mM MgCl2 and 0.5% (vol/vol) NP-40], followed by addition of 1ml of ice cold HB+NS buffer [10 mM Tris-HCl pH 7.5, 10mM NaCl, 2.5mM MgCl2, 0.5% vol/vol, NP-40 and 10% wt/vol sucrose].Cell nuclei were collected by centrifugation at 1,400rpm for 5 minutes at 4°C, resuspended in 125µl of NUN1 buffer [20mM Tris-HCl pH 7.9, 75mM NaCl, 0.5mM EDTA and 50% vol/vol glycerol].After addition of 1.2 ml of ice cold NUN2 buffer [20mM HEPES-KOH pH 7.6, 300mM NaCl, 0.2mM EDTA, 7.5mM MgCl2, 1% vol/vol NP-40 and 1M urea], samples were incubated on ice for 15 minute, mixing by vortexing every 3 minutes. The chromatin pellet was resuspended in HSB buffer [10mM Tris pH 7.5, 500mM NaCl and 10mM MgCl2] and treated with TURBO™ DNase at 37˚C for 15 minutes, followed by Proteinase K for 10 minutes at 37˚C and chromatin-RNA was purified by Trizol. Libraries were prepared using standadr Illumina instruction of TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero for Chromatin RNA seq and TruSeq Stranded mRNA Library Prep  kit for poly A RNA-sequencing.
identifiers.namespace: GEO
identifiers.id: GSM2828284