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| Status |
Public on May 13, 2019 |
| Title |
Old_31_Input |
| Sample type |
SRA |
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| Source name |
Bone marrow
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HSCe (CD34+, CD38-,Lineage-)
donor age: 75
donor sex: F
chip antibody: none
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| Treatment protocol |
Miltenyi MACS magnetic bead purification was used to enrich frozen bone marrow MNC for CD34+ cells. The CD34+ fraction was then stained with CD2-TC, CD3-TC, CD4-TC, CD7-TC, CD8-TC, CD10-TC, CD11b-TC, CD14-TC, CD19-TC, CD20-TC, CD56-TC, and Glycophorin A-TC followed by CD34-APC, CD38-PE-Cy7, CD123-PE, CD45RA-FITC and DAPI. Using a BD FACSAria I, HSCe (DAPI-,Lin-, CD34+, CD38-) were FACS sorted. HSCe were sorted into 1mL IMDM 20% FBS. For H3K4me1, H3K4me3, and H3K27me3, 14,000-20,000 HSCe were used per immunoprecipitation; for H3K27ac, 15,000-35,000 cells were used per immunoprecipitation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Micro-ChIPseq was performed using the True MicroChIP (Diagenode, #C01010130) kit and antibodies that had been validated for specificity and reactivity using the MODified Histone Peptide Array (Active Motif, #13001). The following antibodies were used, 1 g H3K27me3 (Millipore, #07-449, Lot: 21494165), 1 g H3K4me3 (Abcam, #ab8580, Lot: GR164207-1), 0.5 g H3K4me1 (Diagenode, #C15410194, Lot: A1862D), or 0.5 g H3K27ac (Abcam, #ab4729, Lot: GR155970-2).
ChIP-seq libraries were prepared using the V1 MicroPlex Library Preparation kit (Diagenode, #C05010011). For the PCR amplification, a total of 16 amplification cycles was used. Libraries were purified using a 1:1 Ampure bead cleanup and eluted in 16 uL of Tris-HCl ph 8.0. Only libraries with enrichment at targeted sites by QPCR were used for sequencing. Libraries were multiplexed and sequenced using an Illumina Hi-Seq-2500 with 50bp single end sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
O_Pooled_H3K4me1_control_lambda.bdg
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| Data processing |
FastQC was run all samples. Samples with a sequencing duplication rate of >90% were discarded.
Adapters and reads of less than 25 basepairs were removed using cutadapt (version 1.12).
Reads were aligned to the hg19 genome using bowtie2 (version 2.0.5).
Reads were filtered to retain only those that were aligned and had one alignment (!XS:i:N) samtools (version 1.2)
Unique mapped reads for each IP type for each age group were pooled using samtools merge (version 1.2)
Peak calling using the pooled IP and its pooled corresponding Input was performed using macs2 (version 2.0.10.20131216) with the --nomodel option. For H3K4me1, H3K4me3, and H3K27ac, a q-value cutoff of 0.0001 with the narrow peak option was used. Broad peak calling with a q-value cutoff of 0.1 was used for H3K27me3.
Bigwig files of the fold-enrichment(IP/Input) were generated using macs2 SPMR peak calling, followed by macs2 bdgcmp with the -m FE option.
Bigwig files of the log2(IP/Input) were generated using Deeptools2, with the following command: bamCompare --bamfile1 ip --bamfile2 input --outFileName name_log2.bw --ratio log2 -p 30 --verbose --outFileFormat bigwig --scaleFactorsMethod readCount.
Genome_build: hg19
Supplementary_files_format_and_content: One of the processed files is the bedgraph file containing the tag chromosome coordinates and score produced from macs2 peak calling on the pooled IPs and Inputs. For Input samples, the control_lamda.bdg file was uploaded, and for IPs, the treat_pileup.bdg was uploaded. If a donor was used for multiple IP types, then multiple bedgraph files (corresponding to each IP type) are included for the Input. The "_FE.bw" is a bigwig file that contains the fold-enrichment of the IP/Input. The "_log2.bw" is a bigwig file that contains the read-normalized log2(IP/Input) enrichment.
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| Submission date |
Sep 28, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Maria E. Figueroa |
| E-mail(s) |
[email protected]
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| Phone |
305-243-7333
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| Organization name |
University of Miami Miller School Of Medicine
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| Department |
Human Genetics
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| Street address |
1501 NW 10th Ave, BRB 742F
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| City |
Miami |
| State/province |
Florida |
| ZIP/Postal code |
33136 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE104404 |
Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia (ChIP-seq) |
| GSE104408 |
Aging Human Hematopoietic Stem Cells Manifest Profound Epigenetic Reprogramming of Enhancers That May Predispose to Leukemia |
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| Relations |
| BioSample |
SAMN07717802 |
| SRA |
SRX3229626 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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