 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 04, 2024 |
| Title |
ER-Hoxa9_BRQ_96h_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
63.3 mouse GMP cells
|
| Organism |
Mus musculus |
| Characteristics |
treatment: Brequinar 2uM
time point: 96h
|
| Growth protocol |
Cells were grown in RPMI (Corning, 15-040-CV) with 10% dialyzed fetal bovine serum (Sigma, F0392), 4mM glutamine, and 1% penicillin/streptomycin (Corning, 45000-652), supplemented with 0.5uM beta-estradiol (Sigma, E2758; made from a 10mM stock dissolved in 100% ethanol) and stem cell factor (SCF) from conditioned media generated from a Chinese hamster ovary (CHO) cell line that stably secretes SCF (Sykes et al. Cell 2016; this conditioned media was added at a concentration of 1%).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen RNEasy PLUS kit with additional on-column DNAse step. Total RNA is quantified using the Quant-iT™ RiboGreen® RNA Assay Kit and normalized to 5ng/ul. An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet. Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.
An aliquot of 200ng for each sample is transferred into library preparation which is an automated variant of the Illumina TruSeq™ Stranded mRNA Sample Preparation Kit. This method preserves strand orientation of the RNA transcript. It uses oligo dT beads to select mRNA from the total RNA sample. It is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant 500bp cDNA then goes through library preparation (end repair, base ‘A’ addition, adapter ligation, and enrichment) using Broad designed indexed adapters substituted in for multiplexing. After enrichment the libraries were quantified with qPCR using the KAPA Library Quantification Kit for Illumina Sequencing Platforms and then pooled equimolarly. The entire process is in 96-well format and all pipetting is done by either Agilent Bravo or Hamilton Starlet. Pooled libraries are normalized to 2nM and denatured using 0.1 N NaOH prior to sequencing. Flowcell cluster amplification and sequencing were performed according to the manufacturer’s protocols using either the HiSeq 2000 or HiSeq 2500. Each run is a 101bp paired-end with an eight-base index barcode read. Data is analyzed using the Broad Picard Pipeline which includes de-multiplexing and data aggregation.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNAseq
BRQ_96h_rep1
|
| Data processing |
Mouse and human data were mapped to mm10 and hg38 genomes, respectively, using STAR v2.7.8a and default parameters.
Reads that mapped to transcripts were counted using featureCounts v2.0.1 (with parameters -M -O --fraction --primary).
Genome_build: mm10
Genome_build: hg38
Supplementary_files_format_and_content: A tab-delimited file containing raw counts generated from featureCounts.
|
| |
|
| Submission date |
Apr 19, 2021 |
| Last update date |
May 04, 2024 |
| Contact name |
Brian Do |
| E-mail(s) |
bdo311@gmail.com
|
| Organization name |
MIT
|
| Department |
Biology
|
| Lab |
Vander Heiden
|
| Street address |
500 Main St
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE172333 |
A myeloid maturation program initiated by nucleotide depletion during S phase [RNA-Seq] |
| GSE172335 |
Nucleotide depletion promotes cell fate transitions by inducing DNA replication stress |
|
| Relations |
| BioSample |
SAMN18796540 |
| SRA |
SRX10635717 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
