 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 15, 2021 |
| Title |
ucMSC - H3K36me3 - Young - Batch1 |
| Sample type |
SRA |
| |
|
| Source name |
mesenchymal stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: umbilical cord-derived mesenchymal stem cells (PCS-500-010, lot #63216949)
cell passage: PD12
chip antibody: H3K36me3: Active Motif #61101, lot #32412003
|
| Treatment protocol |
Cells were cultured to PD12 and PD32.
|
| Growth protocol |
Human cord blood mesenchymal stem cells (hMSCs) were purchased from ATCC (PCS-500-010, lot #63216949). Cells were grown in low glucose DMEM (Life Technologies 11885-084) supplemented with 10% FBS (Life Technologies 16000-044, lot #1314735) and 1× penicillin/streptomycin (Life Technologies 15140-122) at 37ºC with 5% CO2 and 3% O2. Medium was replaced every 4 days in the absence of passaging. Cells were grown to ~70% confluence and split 1:4 as needed. Cell density was below the concentration needed for reliable detection with a hemocytometer (105 cells/mL), so the growth curve was estimated as 2 population doublings per passage.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 1 million PD12 and PD32 hMSCs were fixed in 1% formaldehyde at room temperature for 10 minutes. Following 3 washes in cold PBS, cell pellets were flash frozen on dry ice and store at -80ºC. Samples were thawed on ice and resuspended in 100uL RIPA with 10uM PFMS and 1× Halt Protease Inhibitor Cocktail (Thermo Fisher PI78439). Samples were sonicated to release chromatin with an average fragment length of 400bp in an EpiShear Multi-Sample sonicator (Active Motif 53062) using the following parameters: 5 minutes sonication, 30 second ON/OFF cycles, 50% amplitude at 4ºC. For each ChIP, 10uL of clarified lysate was added to 90uL of HBSS and used as input into the True MicroChIP Kit (Diagenode, C01010130); manufacturer’s instructions were followed beyond the fixation/sonication part of the protocol, using 2ug of each antibody. 2uL of clarified lysate was used as input and was treated as a sample starting from the reverse crosslinking step in the protocol. The ChIPed DNA was cleaned and concentrated using the Qiaquick PCR Purification Kit (Qiagen, 28104), following the manufacturer’s protocol with the following exception
Sequencing libraries were prepared using the MicroPlex Library Preparation Kit v2 (Diagenode, C05010014) following manufacturer’s instructions
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Raw reads were trimmed to remove sequencing adaptors and low quality reads using Trim Galore version 0.4.4 with default parameters.
ChIP-seq reads were aligned to the hg19 genome assembly using using bowtie2 version 2.2.4 with default parameters.
Duplicated reads were removed using Picard with default parameters.
Peak calling was performed using MUSIC with default parameters, except that H3K9me3, H3K36me3 and H3K27me3 were performed using the get_motimal_broad_ERs model and peaks were called for the remaining datasets using get_optimal_punctate_ERs model.
Differentially bound regions were identified by comparing the changes between young and old data using the csaw R package version 3.11 with H3 total and input as internal control for histone markers and TBP, respectively.
Signal tracks were generated using H3 total or input as control. Deeptools bamCompare version 3.2.0 were used with paraeters: -p max --operation subtract -bs 10 --minMappingQuality 30 --extendReads --ignoreDuplicates --scaleFactors --blackListFileName blacklist.bed. scaleFactors were calucluated using SES method. black list regions of hg19 were downloaded from ENCODE website
Genome_build: hg19
Supplementary_files_format_and_content: normalized signal tracks in bigwig format
Supplementary_files_format_and_content: peak files in bed format
|
| |
|
| Submission date |
Aug 18, 2020 |
| Last update date |
Jul 15, 2021 |
| Contact name |
Weiwei Dang |
| E-mail(s) |
weiwei.dang@bcm.edu
|
| Organization name |
Baylor College of Medicine
|
| Street address |
1 Baylor Plaza
|
| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE156406 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters (ChIP-Seq) |
| GSE156409 |
Age-associated cryptic transcription in mammalian stem cells is linked to permissive chromatin at cryptic promoters |
|
| Relations |
| BioSample |
SAMN15848249 |
| SRA |
SRX8961852 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4730295_H3K36me3_Y.bw |
826.6 Mb |
(ftp)(http) |
BW |
| GSM4730295_H3K36me3_Y_peaks.bed.gz |
295.4 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
