GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM6588310

Query DataSets for GSM6588310

Status

Public on May 23, 2023

Title

LSK-LinPriming_NTC_rep2_batch3

Sample type

SRA

Source name

Bone-Marrow

Organism

Mus musculus

Characteristics

tissue: Bone-Marrow
cell type: Lin- c-kit+ Sca1+
genotype: Cas9-GFP Wild-type
treatment: Ex vivo grown: SCF, Flt3L, Tpo.

Treatment protocol

Cas9-LSK cells were transduced with lentiviral vectors expressing sgRNAs against specific CFs

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)

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.

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

NextSeq 1000

Description

Cas9-LSK progenitors were transduced with Lentiviral vectors expressing sgRNAs targeting specific Chromatin factors
LSK-WT_ATAC3-merged_peaks.broadPeak

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

Submission date

Sep 16, 2022

Last update date

May 23, 2023

Contact name

Brian Huntly

E-mail(s)

bjph2@cam.ac.uk

Organization name

University of Cambridge

Department

Department of Haematology