GSM3325457: CRIS-clone rep2; Homo sapiens; RNA-Seq
Neuroblastoma cell line SK-N-BE(2)C was cultured in 6-well plate for 2 days. Next, the cells were homogenized in 500 μl TRIzol® Reagent and cell lysates were collected. Then 100 μl chloroform was added and cellular RNAs were extracted. The solution was centrifuged at 12,000 xg for 15 minutes. Supernatant was collected into new eppendorf with same volume of 100% EtOH and centrifuged at 13,000 xg for 1 minute. RNA wash buffer (400 μl) was added to the column and centrifuged at 13,000 xg for 1 minute. DNase I (5 μl) and DNA Digestion Buffer (75 μl) was added into column and kept at RT for 15 minutes. Direct-zol RNA Prewash (400 μl) was added to the column and centrifuged at 13,000 xg for 1 minute. Washing step was repeated again. After next, RNA Wash Buffer (700 μl) was added to the column and centrifuged at 13,000 xg for 2.5 minutes. Flow-through was discarded and centrifuged at 13,000 xg for 2.5 minutes again. Then, column was transferred into new RNase-free tube. To elute RNA, RNase-free water (20 μl) was added directly into the column matrix and centrifuged at 13,000 xg for 1 minute. RNA concentration was determined by NanoDrop ND-1000 (NanoDrop Technologies, Montchanin, DE, USA) and RNA quality was checked by 1% agarose gel electrophoresis. Libraries were constructed uing SureSelect Strand Specific RNA Library kit according to the manufacturers
GEO Accession
GSM3325457