 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 22, 2023 |
| Title |
LSC_p63_2 |
| Sample type |
SRA |
| |
|
| Source name |
LSC_aberdam
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LSC_aberdam
cell type: Limbal stem cells
chip antibody: Santa Cruz #H129, 1 μg, recognizing the C-terminal α tail of p63
|
| Growth protocol |
primary KCs were cultured in Keratinocyte Basal Medium supplemented with 100 U/mL Penicillin/Streptomycin, 0.1 mM ethanolamine, 0.1 mM O-phosphoethanolamine, 0.4% bovine pituitary extract, 0.5 μg/mL hydrocortisone, 5 μg/mL insulin and 10 ng/mL epidermal growth factor. Medium was refreshed every other day until the cells were 90% confluent. LSC were cultured in KSFM supplemented with 25 μg/ml Bovine Pituitary Extract, 0.2 ng/ml Epidermal Growth Factor, 0.4 mM CaCl2, 2 mM Glutamine and 100 U/ml Penicillin/Streptomicin. Medium was refreshed every other day untill the cells were 90% confluent.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin was incubated overnight at 4 °C in 1× incubation buffer (0.15% SDS, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES) supplemented with protease inhibitors and 0.1% BSA. A 50:50 mix of Protein A and G Dynabeads (Invitrogen) were added. Antibodies against H3K4me3 (Diagenode, C15410003, 1 μg), TP63 (Santa Cruz #H129, 1 μg, recognizing the C-terminal α tail of p63), H3K27ac (Diagenode #C15410174, 1.2 μg) and H3K27me3 (Diagenode #C15410069, 1.5 μg) were used in each ChIP assay. The beads were washed twice with wash buffer 1 (0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), once with wash buffer 2 (wash buffer 1 with 500 mM NaCl), once with wash buffer 3 (250 mM LiCl, 0.5% sodium deoxycholate, 0.5% NP-50, 1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES), and twice with wash buffer 4 (1 mM EDTA, 0.5 mM EGTA, and 20 mM HEPES). After the washing steps, beads were rotated for 20 min at room temperature in elution buffer (1% SDS and 0.1 M NaHCO3). The supernatant was de-cross-linked with 200 mM NaCl and 100 μg/mL proteinase K for 4 h at 65 °C. De-cross-linked DNA was purified with MinElute PCR Purification columns (Qiagen). DNA amounts were determined with Qubit fluorometric quantification (ThermoFisher Scientific).
The pulled down DNA fragments were proceeded on with library construction using KAPA Hyper Prep Kit (Kapa Biosystems #KK8504) according to the standard protocol. The prepared libraries were then sequenced using the NextSeq 500 (Illumina) according to standard Illumina protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Preprocessing of reads was done automatically with workflow tool seq2science v0.7.1
Paired-end reads were trimmed with fastp v0.20.1 with default options. Genome assembly GRCh38.p13 was downloaded with genomepy 0.11.1. Reads of ChIP-seq were aligned with bwa-mem v0.7.17(Li, 2013)with options '-M'. ChiP sample peaks were called with macs2 v2.2.7(Zhang et al., 2008) with optionsthe options ‘--keep-dup 1 --buffer-size 10000 -q 0.1' in BAM mode for narrow peaks datasets ATAC, H3K4me3, TP63. While for broad peaks datasets H3K27me3 and H3K27ac the options: ‘--keep-dup 1 –broad’ were used. The effective genome size was estimated by taking the number of unique kmers in the assembly of the same length as the average read length for each sample.
Supplementary files format and content: bigWig, narrowPeak (except for Input sample)
|
| |
|
| Submission date |
Jun 24, 2022 |
| Last update date |
Sep 22, 2023 |
| Contact name |
Jo Huiqing Zhou |
| E-mail(s) |
jo.zhou@radboudumc.nl
|
| Organization name |
Radboud University
|
| Street address |
Geert Grooteplein 26/28
|
| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18573 |
| Series (2) |
| GSE206920 |
Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease [ChIP-seq] |
| GSE206924 |
Multi-omics analyses identify transcription factor interplay in corneal epithelial fate determination and disease |
|
| Relations |
| BioSample |
SAMN29336040 |
| SRA |
SRX15891789 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6266883_LSC_p63_2.bw |
141.9 Mb |
(ftp)(http) |
BW |
| GSM6266883_LSC_p63_2_peaks.xls.gz |
318.6 Kb |
(ftp)(http) |
XLS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
