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| Status |
Public on Mar 01, 2016 |
| Title |
H1 |
| Sample type |
SRA |
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| Source name |
human embryonic stem cells, WiCell
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H1
lentiviral transgenes: no
passage number: passage 40
cell type: embronic stem cells
culture protocols: Human ESCs (H1 and H9, WiCell, Wisconsin) and iPSCs were maintained in E8 medium [27] on MatrigelÂcoated tissue culture plates. E8 medium contained DMEM/F12, 64 mg/L LÂascorbic acid 2Âphosphate sesquimagnesium, 13.6 µg /L sodium selenium, 1.7 g/L NaHCO3, 1 g/L sodium chloride, 10 ng/ml FGF2, 20 µg/ml insulin, 10 µg/ml transferrin and 2 µg/L TGFβ1
harvest time: 80% confluence
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| Treatment protocol |
Seed BJ cells into 6-well plate at 100,000 cells/well. Twenty-four hours post plating, add OSK (OCT4, 10 MOI; SOX2, 5 MOI and KLF4, 5 MOI) viruses with or without GFP or BRD3R viruses into respective wells. Incubate overnight. Next morning, remove viruses by replacing virus-containing media with fresh fibroblast media. Seventy-two hour post transduction, harvest RNA.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were harvested with Trizol reagent and stored at -80 °C until use. Total RNA was extracted using the Direct-zolTM Miniprep kit (R2052).
The standard Agilent SureSelect Strand-Specific RNA sequencing protocol was followed. Briefly, the polyA fraction was purified using two rounds of oligo dT-magnetic bead purification. The resulting polyA fraction was fragmented and first strand cDNA was done with random primers in the presence of Actinomycin D using standard techniques. The resulting first stand cDNA was purified using AMPure Beads and second strand cDNA and end repair was done using standard techniques. The blunt ended double stranded cDNA was incubated with exo- E.coli DNA polymerase to add an adenosine to the ends for T/A cloning of the adaptors. PCR amplification was done in the presence of uracil DNA glycosylase to generate strand specificity. A second round of PCR amplification was done to add unique 6-bp barcodes to each sample and add sequences necessary for flow cell attachment. The resulting mRNA libraries were quantitated using qPCR following the manufacturer's instructions (Kapa Biosystems, Woburn MA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalls performed using CASAVA version 1.8.4
Calculating insert size and standard deviation performed using Picard v.1.126
Reads were mapped to hg19 using TopHat v2.0.13 guided by GTF version GRCh37.70 with default parameters and calculated insert size and SD
read counting performed using HT-seq v0.6.0 (--mode=union --stranded=no --minaqual=30 --idattr=gene_name –type=exon)
Normalization and Deferential expression analysis performed using DEseq v3.0
BAM to bedgraph performed using bedtools v2.17.0
bedgraph to bigwig performed using bedGraphToBigWig v4
Statistics and plotting performed in R v3.1.1
Genome_build: hg19
Supplementary_files_format_and_content: BigWig (coverage for mapped reads), count tables (read counts for genomic features) and normalized count tables
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| Submission date |
Mar 11, 2015 |
| Last update date |
Apr 06, 2020 |
| Contact name |
Alireza Khodadadi-Jamayran |
| Organization name |
New York University, NYU Langone Medical Center
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| Department |
Division of Advanced Research Technologies (DART)
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| Lab |
Applied Bioinformatics Laboratories (ABL)
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| Street address |
550 1st Ave, MSB 304
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| City |
New York City |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE66798 |
The acetyllysine reader BRD3R promotes human nuclear reprogramming and regulates mitosis |
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| Relations |
| Reanalyzed by |
GSM4101209 |
| Reanalyzed by |
GSM4455239 |
| BioSample |
SAMN03400454 |
| SRA |
SRX952201 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1632428_H1.bw |
182.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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