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Status |
Public on Nov 29, 2024 |
Title |
CUT&Tag_EGFP-2 |
Sample type |
SRA |
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Source name |
cell line
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Organism |
Homo sapiens |
Characteristics |
tissue: cell line treatment: reprogramming genotype: wild type time: Day10 antibody: anti-EGFP
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Treatment protocol |
Reprogramming intermediate population was collected by magnetic-activated cell sorting (MACS) through Pluripotent Stem Cell MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions.
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Growth protocol |
hiF-T cells were seeded on feeder cells, then cultured in hESM + Doxycycline (Dox, 1 ng/mL) for 6 days, followed by either culturing as before to generate primed iPSCs or switching to 5iLAF medium to generate naive iPSCs.
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Extracted molecule |
genomic DNA |
Extraction protocol |
CUT&Tag was performed by Hyperactive Universal CUT&Tag Assay Kit for Illumina (Vazyme). Briefly, a total of 50,000 cells were harvested, washed by Wash Buffer, and centrifuged for 5 min at 300 × g at room temperature. 10 μL pre-activated ConA beads were added to per sample and incubated at room temperature for 10 min. The supernatant was discarded and the cell-beads complex was resuspended with 50 μL Antibody Buffer with 1 μg primary antibody (Anti-HA, Cell Signaling Technology #3724S; Anti-H3K9me3, Active Motif #39161) and incubated at 4 °C overnight. Then the primary antibody was removed, added 50 μL Dig-wash Buffer with 0.5 μg secondary antibody (Anti-Rb IgG, antibodies-online #ABIN101961), and rotated at room temperature for 1 h. After washing 3 times with 200 μL Dig-wash buffer, 2 μL PA/G–Tnp was added with 98 μL Dig-300 buffer and rotated at room temperature for 1 h, then washed 3 times with 200 μL Dig-wash buffer. 40 μL Dig-300 Buffer was mixed with 10 μL 5 × TTBL, then added and incubated at 37 °C for 1 h. The fragmented DNA was extracted with DNA Extract Beads and then amplified upon the appropriate cycles. Libraries were purified with 2 × AMPure XP (Beckman Coulter). Library concentration was determined by Qubit dsDNA HS Kit (Invitrogen). The resultant CUT&Tag libraries were performed paired-end sequencing on the NovaSeq 6000 platform (Illumina) according to the manufacturer’s instructions at Nanjing Jiangbei New Area.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
*library strategy: CUT&Tag Sequencing reads were first trimmed to remove adapters and then mapped to the annotated human transcripts (UCSC hg19) using bowtie2 (v2.3.5.1) with the parameters:-q --no-mixed --no-discordant --no-unal. High confidence reads were kept for further analysis using SAMtools (v1.7) with the parameters: -bf 0x2 -q 30. PCR duplicate reads removed using sambamba (v0.7.0). Genome-wide signals were calculated using a 25-bp window and normalized to the uniquely mapped fragments using ‘bamCoverage’ from deepTools (v2.5.7) Peak calling was performed using macs2 (v2.1.2). Assembly: hg19 Supplementary files format and content: tab-delimited peak files include protein binding sites
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Submission date |
Feb 15, 2024 |
Last update date |
Nov 29, 2024 |
Contact name |
Liping Wang |
E-mail(s) |
wanglp13@gmail.com
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Organization name |
Tongji University
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Street address |
1239 Siping Road
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City |
Shanghai |
ZIP/Postal code |
200082 |
Country |
China |
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Platform ID |
GPL24676 |
Series (2) |
GSE255860 |
The divergent role of two PRDM1 isoforms during human naïve pluripotency reprogramming [CUT&Tag] |
GSE255862 |
The divergent role of two PRDM1 isoforms during human naïve pluripotency reprogramming |
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Relations |
BioSample |
SAMN39954611 |
SRA |
SRX23635823 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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