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| Status |
Public on Feb 01, 2023 |
| Title |
vcAlkbh5_unlab |
| Sample type |
SRA |
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| Source name |
lineage-depleted bone marrow
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| Organism |
Mus musculus |
| Characteristics |
strain: Vav-iCre+; Alkbh5fl/fl
agent: None
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| Treatment protocol |
Lineage-depleted bone marrow cells were treated with 100 µM s4U for 2 hours.
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| Growth protocol |
Mouse bone marrow cells were lineage depleted and grown in Dulbecco’s Modified Essential Media supplemented with 10% fetal bovine serum (Gibco) 1x L-glutamine, 50 ng/ml mSCF, 50 ng/mL mTPO, 50 ng/mL mFlt3L, 10 ng/mL mIL3. Cell cultures were maintained at 37℃.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was harvested with Trizol reagent, followed by chloroform extraction and isopropanol precipitation. For TimeLapse-seq (TL-seq) genomic DNA was depleted using Turbo DNAse, followed by cleanup with RNAclean beads. RNA was treated with oxidative-nucleophilic-aromatic-substitution chemistry as described in Schofield et al. Nature Methods 2018.
TL-seq libraries were generated following standard protocols using the SMARTer Stranded Total RNA-Seq Kit-Pico Input Mammalian, V2 (Takara Bio USA, cat. 634413).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Reads were filtered for unique sequences using FastUniq
Reads were trimmed using cutadapt (--minimum-length=20)
Reads were aligned to the mm10 genome using HISAT2 aligned with default parameters and --mp 4,2 .
Reads were processed using Picard tools, including FixMateInformation, SortSam and BuildBamIndex
Reads were filtered for those that aligned uniquely (flag: 83/163, 99/147), with MAPQ ≥ 2, and without insertions.
HTSeq-count was used to identify mapped reads in UCSC transcripts (union mode)
T-to-C mutations were identified using Rsamtools and a custom R script (available on Bitbucket). Only mutations at positions with a base quality score of greater than 40 that were at least three nt from the end of the read were counted. Reads were excluded where there were greater than five T-to-C mutations and these mutations did not account for at least one third of the observed mutations (NM tag).
SNP sites were filtered from our data in two ways: 1, bcftools was used to identify T-to-C SNP sites in control samples, and these sites were excluded from analysis; 2, sites exhibiting high T-to-C mutation rates in non-s4U treated controls were excluded from analysis.
To examine the distribution of reads with each minimum number of T-to-C mutations, bam files were filtered using Picard tools.
STAR aligner was used to make genome-coverage tracks (inputAlignmentsFromBam mode, outWigType bedGraph), and tracks were normalized using DESeq2 (estimateSizeFactors).
Tracks were converted to binary format using IGVtools (toTDF).
Genome_build: GRCm38
Supplementary_files_format_and_content: Bedgraph files where reads are separated by the number of T-to-C mutations (0-5+) and strandedness (min/pos)
Supplementary_files_format_and_content: Comma-separated values for raw read counts by gene
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| Submission date |
Jan 21, 2022 |
| Last update date |
Feb 02, 2023 |
| Contact name |
Matthew Simon |
| E-mail(s) |
matthew.simon@yale.edu
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| Organization name |
Yale University
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| Street address |
300 Heffernan Dr, 300 Hefferna Dr
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| City |
West Haven |
| State/province |
Connecticut |
| ZIP/Postal code |
06516 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE194148 |
TimeLapse-seq reveals that ALKBH5 modulates RNA stability of metabolic transcripts |
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| Relations |
| BioSample |
SAMN25153975 |
| SRA |
SRX13853411 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5829499_vcALKBH5_unlab_counts.csv.gz |
203.7 Kb |
(ftp)(http) |
CSV |
| GSM5829499_vcAlkbh5_unlab.TC.0.min.bedGraph.gz |
94.3 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.0.pos.bedGraph.gz |
95.7 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.1.min.bedGraph.gz |
6.8 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.1.pos.bedGraph.gz |
6.9 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.2.min.bedGraph.gz |
275.4 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.2.pos.bedGraph.gz |
309.6 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.3.min.bedGraph.gz |
11.1 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.3.pos.bedGraph.gz |
17.2 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.4.min.bedGraph.gz |
1.1 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.4.pos.bedGraph.gz |
1.7 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.5.min.bedGraph.gz |
369 b |
(ftp)(http) |
BEDGRAPH |
| GSM5829499_vcAlkbh5_unlab.TC.5.pos.bedGraph.gz |
452 b |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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