GSM8172227: 3T3_CDX2, RNA, noDox, rep1; Mus musculus; RNA-Seq
500,000 NIH-3T3 iCDX2 cells were treated for 6 hours with 500ng/ml Doxycycline or solvent alone prior to cell collection. Cell pellets were resuspended in 500 μlL Nuclear RNA lysis buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mMMgCl2, 1% BSA (Merck, B9000S), 0.1% Tween (Fisher Scientific, 10113103), 1 mM DTT (Merck, 646563), 0.5U/μl Protector RNase Inhibitor (Merck, 3335399001), 0.1% NP-40, 0.01% Digitonin (Invitrogen, BN2006)) and incubated 5 min on ice. Then 500 μl Nuclear RNA wash buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA (Merck, B9000S), 0.1% Tween (Fisher Scientific, 10113103), 1 mM DTT (Merck, 646563), 0.5U/μl Protector RNase Inhibitor (Merck, 3335402001) were added before spinning down cells at 300g 4°C for 5 min. Two more washes with Nuclear RNA wash buffer were performed before proceeding to RNA extraction using the Rneasy Plus Micro Kit (Qiagen,74034) with the addition of an on-column DNAse digestion (Qiagen, 79254)MgCl2, 1% BSA (Merck, B9000S), 0.1% Tween (Fisher Scientific, 10113103), 1 mM DTT (Merck, 646563), 0.5U/μl Protector RNase Inhibitor (Merck, 3335399001), 0.1% NP-40, 0.01% Digitonin (Invitrogen, BN2006)) and incubated 5 min on ice. Then 500 μl Nuclear RNA wash buffer (10 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA (Merck, B9000S), 0.1% Tween (Fisher Scientific, 10113103), 1 mM DTT (Merck, 646563), 0.5U/μl Protector RNase Inhibitor (Merck, 3335402001) were added before spinning down cells at 300g 4°C for 5 min. Two more washes with Nuclear RNA wash buffer were performed before proceeding to RNA extraction using the Rneasy Plus Micro Kit (Qiagen,74034) with the addition of an on-column DNAse digestion (Qiagen, 79254) NEBNext Ultra II Directional RNA Library Prep Kit for Illumina