GSM9159085: BJ_OSK_LucSHRNA1; Homo sapiens; RNA-Seq
BJ fibroblast cells (ATCC, CRL-2522, foreskin fibroblasts) were cultured in six well plates at a density of 2 00000 cells per well. At 24 hrs post seeding, the medium was removed and 2 mL fresh medium containing polybrene (final concentration = 5 µg/mL) was added to the wells. The viruse preparations were then added into respective wells (day 0). At 18 hrs after virus addition, the virus-containing spent media were removed, and fresh fibroblast medium was added. At 72 hrs (or indicated times) after virus removal (on Day 4), the spent medium was removed and 1 mL of TRIzol (Invitrogen; Cat no. 15596018) was added directly to each well. RNA was extracted following manufacturer's protocol. After extraction, the RNA was further purified using Quick RNA Miniprep kit (Zymo Research; Cat. no. R1055) using manufacturer's protocol.   RNA for the nontreated BJ was extracted the same way but without virus transduction. RNA from humna iPSCs or ESCs was prepared using the same kits. Human PSCs were grown in E8 medium Briefly, mRNA from BJ or human PSCs is purified from total RNA using poly-T oligo-attached magnetic beads. The mRNA is first fragmented randomly by the addition of fragmentation buffer. The Novogene ENB library protocol was used as briefed below (from Novogene). ENB library: Then first strand cDNA is synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis is subsequently performed using DNA Polymerase I and RNase H. Double-stranded cDNA is purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA are converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure is ligated to prepare for hybridization (1). In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments are purified with AMPure XP system (Beckman Coulter, Beverly, USA). Finally, the final library is gotten by PCR amplification and purification of PCR products by AMPure XP beads.