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Sample GSM8081801 Query DataSets for GSM8081801
Status Public on Nov 29, 2024
Title CUT&Tag_EGFP-2
Sample type SRA
 
Source name cell line
Organism Homo sapiens
Characteristics tissue: cell line
treatment: reprogramming
genotype: wild type
time: Day10
antibody: anti-EGFP
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.
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.
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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
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
 
Submission date Feb 15, 2024
Last update date Nov 29, 2024
Contact name Liping Wang
E-mail(s) wanglp13@gmail.com
Organization name Tongji University
Street address 1239 Siping Road
City Shanghai
ZIP/Postal code 200082
Country China
 
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
Relations
BioSample SAMN39954611
SRA SRX23635823

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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