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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 28, 2021 |
| Title |
Cut & Run LSK IgG |
| Sample type |
SRA |
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| Source name |
LSK
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| Organism |
Mus musculus |
| Characteristics |
genotype: WT
strain: C57BL/6
harvest condition: 6 days post deletion
antibody: IgG, Cell Sinaling (#2729S)
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| Treatment protocol |
pI;pC was injected to induce deletion in the mice. Cells were sorted for use without any culture or other treatment
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| Growth protocol |
The cells were sorted from mice with different genotypes and directly used for prepareing libraries.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Trizol was used for RNA seq extraction.
RNA libraries were prepared for sequencing using standard Illumina protocols, ATAC seq library was based on Illumina Tagment DNA Enzyme and Buffer Kit. Cut&Run was followed as published online protocols
RNA-Seq, ATAC seq and Cut &Run libraries werer prepared with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina from New England Biolabs
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Sequencing reads in FASTQ format were first subjected to quality control to assess the need for trimming of adapter sequences or bad quality segments. The programs used in these steps were FastQC v0.11.7, Trim Galore! v0.4.2 and cutadapt v1.9.1. The trimmed reads were aligned to the Mus musculus reference genome version GRCm38/mm10 with the program STAR v2.6.1e for RNA-seq and with BOWTIE v2.3.4.1 for ATAC-seq and CUT&RUN-seq experiments. Aligned reads were stripped of duplicate reads with the program sambamba v0.6.8.
For RNA seq, Gene-level expression was assessed by counting features for each gene, as defined in the NCBI's RefSeq database. Read counting was done with the program feature Counts v1.6.2 from the Rsubread package. Raw counts were normalized as transcripts per million (TPM). Differential gene expressions between groups of samples were assessed with the R package DESeq2 v1.26.0.
For ATAC seq, Peaks were called with MACS2 v2.1.2 using the --broad option. ATAC genomic regions among all samples were obtained by merging called peaks at 50% overlap using BEDtools v2.27.0 and in-house programs. This was done at two levels using, between replicates and between treatments. In the former, we require peaks to be present in all biological replicates, meaning a peak observed in one replicate has to have a peak present in all other replicates at the 50% cutoff. On the latter, we keep all peaks from different treatments (merging the peaks in common at 50%). The resulting set of peaks, originally in BED format, were converted to a Gene Transfer Format (GTF) to enable fast counting of reads under the peaks with the program featureCounts v1.6.2 (Rsubread package). Each feature in the GTF file has the value "peak" on the second column.
For Cut & Run, reads with an aligned length between 0 and 280bp were selected for peak calling. The cutoff was chosen to include the first peak in the histogram of read lengths between aligned read pairs. Aligned length is defined as the distance in base-pairs from beginning of alignment in read1 to end of alignment in read2. Peak calling was done with the program MACS v2.1.2 using the narrow peaks mode (default). CUT&RUN genomic regions were obtained using the same method used in the analysis of ATAC-seq data. Resulting peaks, originally in BED format, were converted to a Gene Transfer Format (GTF) to enable fast counting of reads under the peaks with the program featureCounts v1.6.2 (Rsubread package).
Genome_build: mm10
Supplementary_files_format_and_content: TPM expression files for RNA seq data and BigWig for ATAC seq and Cut&Run data.
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| Submission date |
Nov 17, 2020 |
| Last update date |
Jul 28, 2021 |
| Contact name |
Zhaowei Tu |
| E-mail(s) |
ZHAOWEI.TU@cchmc.org
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| Organization name |
Cincinnati Children's Medical Center
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| Street address |
3333 Burnet Ave
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| City |
Cincinnati |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE161613 |
Next Generation Sequencing Analysis of Wild Type and Chd8-/- LSK in Transcriptomes, Chromatin accessibility and chromatin state mapping |
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| Relations |
| BioSample |
SAMN16816208 |
| SRA |
SRX9520179 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4910817_LSKIgG_0-280.bw |
27.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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