GSM5261345: KD2_rep3; Homo sapiens; RNA-Seq
RNA from the different cell lines was extracted using 500 µl TRIzol Reagent (Ambion) directly on 10-cm dishes (cell confluency ∼80%, ∼1,000,000 cells). Lysed cells in TRIzol were scraped into tubes, extracted twice with chloroform, the RNA was precipitated with 0.7 volume isopropanol after addition of 20 µg glycogen, and resuspended in UltraPure distilled water. RNA concentration and quality were determined by Nanodrop (ratio 260/230 and 260/280 above 1.8) and Agilent 2100 BioAnalyzer (RIN above 8). Total RNA was used for northern blot analysis, RiboMethSeq, and RT-PCR and RNase H analysis. For deep sequencing, total RNA was prepared from 3 separate dishes for each sample and shipped to Novogene Corporation Inc. (Sacramento, CA) for cDNA library preparation (250~300 bp inserts) and Illumina sequencing (PE150). The RNA was prepared and sequenced one year apart in the USA and in China in two batches, one for the P1, KD1a, and KD1b cells and one for the P2 and KD2 cells.
GEO
GSM5261345