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| Status |
Public on Jan 22, 2019 |
| Title |
GMP6_Tet2KO |
| Sample type |
SRA |
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| Source name |
Hematopoietic cells sorted from bone marrow
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| Organism |
Mus musculus |
| Characteristics |
cell type: Granulocyte-Monocyte Progenitors (GMPs), Lin-cKit+Sca1-CD150-CD16/32+
genotype: Tet2-/-
transgene: No transgene
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| Growth protocol |
Embryonic stem cells were grown in gelation-coated plates in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen Cat. No. 41966) supplemented with 15% fetal bovine serum (FBS, Hyclone), 2 mM Glutamax (GIBCO), 0.1 mM 2-Mercaptoethanol (GIBCO), 1x non-essential amino acids (GIBCO), 1x Pen-Strep (GIBCO), 3μM GSK-3 inhihibtor (CHIR99021), 1 μM MEK1 inhibitor (PD0325901) and leukemia inhibitory factor (LIF, lab made). Acute myeloid leukemia cells were cultured in suspension in non-tissue culture treated plasticware in StemPro-34 SFM media (ThermoFisher Scientific, Cat: 10639011) supplemented with 2mM GlutaMAX (GIBCO), 1X pen/strep (GIBCO), 0.1mM 2-mercaptoethanol (SIGMA), as well as the cytokines SCF (50ng/ml), IL-3 (10ng/ml), and IL-6 (10ng/ml) (PEPROTECH).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq libraries were generated as described previously (Buenrostro et al. 2013; Lara-Astiaso et al. 2014). Briefly, 10,000 freshly isolated cells (MPP, GMP, AML, and ES) were sorted by FACS into ice-cold FACS buffer (PBS + 2%FBS). The cells were pelleted using a swinging bucket centrifuge (500 x g, 10min, 4oC) with settings for low acceleration/deceleration and washed once in ice-cold PBS. The cell pellets were resuspended in 50μl lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% Igepal CA-630) by gentle pipetting and immediately centrifuged one additional time (500 x g, 10min, 4oC). The supernatant was discarded and the pellet containing released nuclei were resuspended gently in 25μl 1xTD buffer containing 1.25μl Tn5 transposase (Nextera sample preparation kit, Illumina). The transposition reaction was allowed to proceed for 45min at 37oC whereafter DNA fragments were isolated using MinElute PCR purification columns (Qiagen) according to manufacturer’s instruction.
To generate multiplex libraries, the transposed DNA was initially amplified for 5x PCR cycles using 2.5μl each of dual-index primers (Nextera index kit, Illumina) and 2.5μl PCR primer cocktail (PPC, Illumina) in a 25μl reaction volume of 1x KAPA HiFi hot-start ready-mix (Kapa BioSystems). The hot-start polymerase was activated prior to adding to the reaction mix by performing a brief pre-incubation step of 3min at 95oC. The amplified fragments were size-selected with AMPure XP beads (0.5X) to remove fragments larger than 600bp and an aliquot was quantified to determine the optimal PCR cycle number to obtain 1/3 of maximum fluorescence intensity (Library quantification kit, Kapa Biosystems). Finally, PCR amplification was performed using the optimal number of cycles determined for each library (max. 18 cycles in total), size-selected with AMPure XP beads (0.5X) and eluted in resuspension buffer (Illumina). The size distribution of the libraries was evaluated on Bioanalyzer (Agilent) and sequenced on NextSeq 550 (Illumina) using 150bp or 75bp paired-end sequencing.
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
DNA, fragmented and adaptor ligated by Tn5 transposase
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| Data processing |
Trimming of the adapters with Trimmomatic v0.33 with ILLUMINACLIP:NexteraPE-PE.fa:1:10:4:1:true TRAILING:3 MINLEN:20 parameters.
Aligned to mm10 genome using Bowtie2 v2.2.9 with -X 2000 -t –very-sensetive parameters.
Removed reads with MAPQ quality < 10 using samtools and awk scripts.
Removed mitochondrial reads using samtools and awk scripts.
Read start sites are adjusted +4/-5 using samtools and awk scripts.
Using PicardTools v2.9.0 sorted by coordinate and removed duplicated reads.
Removed indels with CIGAR “ID” using samtools and awk scripts.
Bam files are transfromed to bigwig files using Biconductor package GenomicAlignments coverage() function.
Genome_build: mm10
Supplementary_files_format_and_content: Binary bigwig files containing trimmed, mapped and de-duplicated reads (mm10)
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| Submission date |
Jun 18, 2018 |
| Last update date |
Jan 22, 2019 |
| Contact name |
Kasper Dindler Rasmussen |
| E-mail(s) |
[email protected]
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| Organization name |
School of Life Sciences, University of Dundee
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| Department |
Division of Molecular, Cellular, and Developmental Biology (MCDB)
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| Street address |
Dow Street
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| City |
Dundee |
| ZIP/Postal code |
DD1 5EH |
| Country |
United Kingdom |
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| Platform ID |
GPL21626 |
| Series (2) |
| GSE115962 |
TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells [ATAC-Seq] |
| GSE115972 |
TET2 binding to enhancers facilitates transcription factor recruitment in hematopoietic cells |
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| Relations |
| BioSample |
SAMN09444847 |
| SRA |
SRX4232606 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3196044_GMP.KO.2.final.sort.fixed.bigwig |
133.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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