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| Status |
Public on Jan 30, 2023 |
| Title |
OCIAML3_PRO_VTP_48h_REP2_211124_GTGAAA_S10 |
| Sample type |
SRA |
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| Source name |
Cell line OCI-AML3
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| Organism |
Homo sapiens |
| Characteristics |
cell line: OCI-AML3
timepoint: 48h
treatment: 330nM VTP50469
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| Treatment protocol |
Treatment regimens indicated for each sample.
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| Growth protocol |
Human cell lines were cultured in RPMI with 10% FBS.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
10 million cells were harvested and washed once with ice cold PBS, cell pellets were resuspended in 250uL wash buffer to get single-cell suspensions. 10mL permealization buffer was added and samples were incubated on ice for 5 min. Cells were spun down and pellets were washed in 10mL wash buffer. Cells were spun again and resuspended in freezing buffer. Cells were counted and permealization was visualized using tryphan blue. 5 million permealized cells were snap-frozen in 500ul freezing buffer in liquid nitrogen and stored at -80°C. All subsequent steps were performed by the Nascent Transcriptomics Core at Harvard Medical School, Boston, MA. In brief, run-on reactions were performed at 37°C. Adenylated 3' adapter was prepared using the 5' DNA adenylation kit (NEB) and ligated using T4 RNA ligase 2, truncated KQ (NEB, per manufacturers instructions with 15% PEG-8000 final) and incubated at 16°C overnight. 180uL of betaine blocking buffer with 0.6 uM blocking oligo was mixed with ligations and incubated for 5 min at 65°C and 2 min on ice prior to addition of streptavidin beads. After T4 polynucleotide kinase (NEB) treatment, beads were washed once each with high salt, low salt, and blocking oligo wash solutions and resuspended in 5' adapter mix.
Eluted cDNA was amplified 5-cycles (NEBNext Ultra II Q5 master mix (NEB)) with Illumina TruSeq PCR primers RP-1 and RPI-X following the manufacturer's suggested cycling protocol for library construction. A portion of preCR was serially diluted and for test amplification to determine optimal amplification of final libraries.
Illumina Next Gen Sequencing NextSeq platform (Illumina, San Diego, CA) was used.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: PRO-seq
FASTQ read pairs were trimmed to 41bp per mate, and read pairs with a minimum average base quality score of 20 retained. Read pairs were further trimmed using cutadapt 1.14 to remove adapter sequences and low-quality 3’ bases (--match-read-wildcards -m 20 -q 10).
R1 reads, corresponding to RNA 3’ ends, were then aligned to the spiked in Drosophila genome index (dm3) using Bowtie 1.2.2 (-v 2 -p 6 --best --un), with those reads not mapping to the spike genome serving as input to the primary genome alignment step (using Bowtie 1.2.2 options -v 2 --best). Reads mapping to the hg38 reference genome were then sorted, via samtools 1.3.1 (-n), and subsequently converted to bedGraph format using a custom script (bowtie2stdBedGraph.pl).
For promoter reads, annotated transcription start sites were obtained from human (GRCh38.99) GTFs from Ensembl. After removing transcripts with {immunoglobulin, Mt, Mt_tRNA, rRNA} biotypes, PRO-seq signal in each sample was calculated in the window from the annotated TSS to +150 nt downstream, using a custom script, make_heatmap.pl.
For differential expression analysis, reads were summed within the TSS to TES window for each active gene. DEseq2, using the Wald test, was used to determine statistically significant differentially expressed genes. Unless otherwise noted, the default size factors determined by DEseq2 were used.
Genome_build: GRCh38/hg38
Supplementary_files_format_and_content: Differential expression (Excel .xlsx format)
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| Submission date |
Feb 10, 2022 |
| Last update date |
Jan 30, 2023 |
| Contact name |
Charlie Hatton |
| E-mail(s) |
Charles_Hatton@dfci.harvard.edu
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| Organization name |
Dana-Farber Cancer Institute
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| Department |
Pediatric Oncology
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| Lab |
Scott Armstrong/CPCT
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| Street address |
450 Brookline Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE196478 |
Mutant NPM1 binds chromatin and directly regulates oncogenic transcription in acute myeloid leukemia [PRO-seq] |
| GSE196480 |
Mutant NPM1 binds chromatin and directly regulates oncogenic transcription in acute myeloid leukemia |
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| Relations |
| BioSample |
SAMN25829572 |
| SRA |
SRX14127338 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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