title: GSM6164734: BJ, OSK, BRD3RDBD1, replicate b; Homo sapiens; RNA-Seq
library_construction_protocol: BJ fibroblast cells (ATCC, CRL-2522, foreskin fibroblasts) were cultured in six well plates at a density of 2 ´ 105 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 after virus removal (on Day 4), the spent medium was removed and 1 mL of TRIzol (Invitrogen; Cat no. 15596018) was added directly to the 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. Human mRNAs are captured via oligo(dT) beads. Non-stranded protocol was used. The purified mRNAs are fragmented randomly in fragmentation buffer and the first-strand cDNA is synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). The second strand is subsequently generated by dNTPs, DNA polymerase I and RNase H. Double-stranded cDNA molecules are purified by AMPure XP beads and overhanging ends are repaired to blunt ends by exonuclease/polymerase. After 5' phosphorylation and 3' adenylation, the cDNAs are ligated with P5/P7 sequencing adapters to prepare for hybridization. In order to select the insert fragment of preferentially 150~200 bp in length, the modified libraries are purified with AMPure XP system (Beckman Coulter, Beverly, USA). The final library is ready after PCR amplification and products purification by AMPure XP beads. The following samples were sequenced using DNA nanoball (DNB) technology, BJ48hS1, BJ48hS2, BJ48HS3, hESH1, hESH9, H1P43, H9P69S2.  mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. The obtained mRNA molecules were fragmented into small pieces using fragmentation reagent. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3' adenylated. Adapters were ligated to the ends of these 3' adenylated cDNA fragments. The cDNA fragments were amplified  with adapters from previous step. PCR products were purified with Ampure XP Beads (AGENCOURT), and dissolved in EB solution. Library was validated on the Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and paired end 100 bases reads were generated in the way of sequenced by synthesis.