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Status |
Public on May 23, 2023 |
Title |
LSK-Mye_Rcor1_rep2_batch2 |
Sample type |
SRA |
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Source name |
Bone-Marrow
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Organism |
Mus musculus |
Characteristics |
tissue: Bone-Marrow cell type: Lin- c-kit+ Sca1+ genotype: Cas9-GFP Rcor1-KO treatment: Ex vivo grown: Flt3L, GM-CSF, IL3, IL6
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Treatment protocol |
Cas9-LSK cells were transduced with lentiviral vectors expressing sgRNAs against specific CFs
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Growth protocol |
Cas9-LSK progenitors were sorted and grown in Lineage Priming Medium (DMEM/F12 100 ng/ml Tpo, 10 ng/ml SCF, 1 ng/ml Flt3L, 1 mg/mL PVA 87%, ITSX 1X, 10 mM Hepes) or Myeloid differentiaton media (20% FBS, 10 ng/ml GM-CSF, SCF, 5 ng/mL G-CSF, IL3, IL6, IL5, Flt3L, 2 ng/mL Tpo and 2 U/mL Epo)
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cells expressing sgRNAs (BFP+) were FACS sorted in 1X PBS + 0.05% BSA, pelleted and permeabilized in 1X TD buffer containing 2 ul Tn5 enzyme (Illumina), 0.01% Digtonin in DMSO, 0.1% Tween 20, 0.1% IGEPAL in Molecular Biology Grade water Omni-ATAC protocol: Tagmentation was performed at 37 ºC for 30 minutes with agitation at 1000 rpm, then stopped by adding EDTA 5 mM final concentration. Next, 2 uL of Proteinase K were added and the samples were incubated for 30 minutes at 40 ºC. Genomic DNA was purified with a 2X SPRI cleanup and tagmented sites were PCR-amplified with 25 uL Kappa HIFI 2X and 2.5 uL of 10 uM P5-i5-read1 and P7-i7-Read2 primers. The PCR cycling conditions are: 5 min at 72 ºC, 10 x [2 min at 98 ºC, 20 s at 98 ºC, 30s at 60 ºC, 1 minute at 72 ºC] 3 min at 72 ºC. Amplified ATAC-seq libraries were purified with a 1.6X SPRI Cleanup, quantified using the Qubit dsDNA high sensitivity assay kit, and then analysed on a Tape Station 4000. ATAC-seq libraries were sequenced at 50 million reads on a NextSeq 1000.
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Library strategy |
ATAC-seq |
Library source |
genomic |
Library selection |
other |
Instrument model |
NextSeq 1000 |
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Description |
Cas9-LSK progenitors were transduced with Lentiviral vectors expressing sgRNAs targeting specific Chromatin factors Mye-Rcor1_ATAC5-merged_peaks.broadPeak
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Data processing |
We followed the ATAC-seq nf-core pipeline ATAC-seq reads were trimmed with default parameters using Trim Galore v0.6.6 with Cutadapt v3.4 ATAC-seq reads were aligned to the GRCm38/mm10 reference genome assembly using Bowtie version 2.3.4.2 with parameters -X 1000 --no-discordant --no-mixed --very-sensitive we removed duplicated regions with Picard v2.25.4 and filtered out ENCODE blacklist regions v2.0 and non-interesting chromosomes We removed Tn5 adaptors with alignmentSieve v3.5.1 We pooled replicates and converted the paired BAM files to single-read bed format using function bamToBed from BEDTools v2.27.1 Peaks were called uing MACS v2.2.7.1 with parameters –broad -f BED --keep-dup all --nomodel --shift -75 --extsize 150 Assembly: GRCm38/mm10 Supplementary files format and content: bamfile, broadPeak
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Submission date |
Sep 16, 2022 |
Last update date |
May 23, 2023 |
Contact name |
Brian Huntly |
E-mail(s) |
bjph2@cam.ac.uk
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Organization name |
University of Cambridge
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Department |
Department of Haematology
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Street address |
Hills Road
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City |
Cambridge |
ZIP/Postal code |
CB2 0AW |
Country |
United Kingdom |
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Platform ID |
GPL32159 |
Series (2) |
GSE213506 |
Comparative binding patterns of Chromatin Regulators in normal versus leukemic hematopoiesis [ATAC-Seq] |
GSE213513 |
Leukemic hematopoiesis |
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Relations |
BioSample |
SAMN30889367 |
SRA |
SRX17605358 |
Supplementary data files not provided |
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Raw data are available in SRA |
Processed data are available on Series record |
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