title: GSM7321040: 3-day 10ng/mL dox-treated Xist-RMCE clone 4 RNA; Mus musculus; RNA-Seq
library_construction_protocol: RMCE ESCs or MEF feeder-free TSCs were washed twice with ice-cold PBS and harvested with 1 mL TRIzol (Thermo Fisher 15596026). Total RNA was prepared via standard TRIzol-chlorofom extraction, per manufacturer's instructions, except for the addition of 4μL 5μg/μL linear acrylamide (Thermo Fisher AM9520) to promote precipitation in isopropanol. RNA pellets were fully resuspended in RNase-free water, RNA concentrations were measured via Qubit 2.0 fluorometer (RNA high sensitivity kit, Thermo Fisher Q32852), and integrity of RNA was confirmed by visualizing rRNA bands on an agarose gel. Working stocks of 100ng/μL total RNA were prepared, and all RNA samples were stored at -80°C. Libraries for RNA sequencing were prepared using the KAPA RNA HyperPrep Kit with RiboErase (HMR) (Roche 08098140702) and KAPA Unique Dual-Indexed Adapters (Roche 08861919702) or TruSeq adapters, with each library's input consisting of 9μL 100ng/μL total RNA (900 ng) mixed with 1μL of a 1:250 dilution of ERCC RNA spike-in controls (Thermo Fisher 4456740). RNA fragmentation was performed at 94°C for 6min. Libraries were pooled and sequenced on an Illumina NextSeq500 platform with a 75-cycle NextSeq 500/550 High Output Kit v2.5 (Illumina 20024906).