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Sample GSM5578008 Query DataSets for GSM5578008
Status Public on Mar 23, 2022
Title CUTRUN_NFIA_Primary_Perpheral Blood_Invitrogen#535936
Sample type SRA
Source name Primary_Peripheral Blood
Organism Homo sapiens
Characteristics differentiation: D11
antibody: NFIA
biological replicate: NA
treatment: no
tissue: Peripheral Blood
Treatment protocol We established the Cas12a stably expressing HUDEP-2 using pRG232 (#149723, Addgene) and puromycin selection. Control and NFIA&NFIX Cas12a gRNA oligos were subcloned into lentiviral pRG212 (#149722, Addgene) using BsmBI restriction site, and then introduced into HUDEP2 cells by lentivirual infection
Growth protocol HUDEP2 Cells were maintained in a medium containing StemSpan SFEM (9650, StemCell Technologies) supplemented with 50 ng/mL recombinant human stem cell factor (300-07, Peprotech), 3 IU/mL Epoetin alfa (Epogen, Amgen), 0.4 μg/mL dexamethasone (D9891, Sigma Aldrich), 1 μg/mL doxycycline (D9891, Sigma Aldrich) and 1% Pen-Strep (15140122, Invitrogen). Erythroid differentiation was induced by placing 2-5 million HUDEP-2 cells into 1x Iscove’s modified Dulbecco’s medium (IMDM, MT10016CV, Mediatech) supplemented with 1% L-glutamine, 330 μg/mL human holo-transferrin (T4132, Sigma Aldrich), 10 μg/mL recombinant human insulin solution (I9278, Sigma Aldrich), 2 IU/mL heparin, 5% inactivated fetal biovine serum (FBS), 3 IU/mL Epoetin alfa (Epogen, Amgen), 1 μg/mL doxycycline and 1% Pen-Strep.
Extracted molecule genomic DNA
Extraction protocol 0.5-1 million undifferentiated HUDEP2, HUDEP2 undergoing 2 days of differentiation, and primary cells undergoing 8 days of erythroid differentiation were used for CUT&RUN. We followed the experimental procedure published by Henikoff lab ( with adaptations15. Antibody incubation was performed in 300μl PCR tubes for 2 hours in the cold room. pA/G-MNase and Spike-In DNA (#40366) were obtained from Cell Signaling. The following antibodies were used: IgG (#3900, Cell signaling), BCL11A (ab191401, Abcam), NFIA (PA5-52252, Thermo Fisher Scientific), NFIX (SAB1401263, Sigma Aldrich), GATA1(Ab11852, Abcam), H3K27Ac (MA5-23516, Invitrogen). All antibodies were used approximately 1μg in 100μl.
The concentration of the released DNA fragments was determined by Qubit and 15-30ng DNA was used for adapter ligation and library construction. The sequencing libraries were generated using the kit from NEB (Cat# E7645 and E6440S). We followed the experimental procedure posted by Nan Liu ( to size select the <120 bp fragment and facilitate the transcription faction binding site identification20. The multiplexed sequencing libraries were pair-end (2x50bp) sequenced on Illumina Nextseq 2000 platform.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model NextSeq 2000
Data processing Library strategy: CUT&RUN-seq
CUT&RUN sequencing reads were analyzed by CUT-RUNTOOLS-2.0 (
Genome_build: hg38
Supplementary_files_format_and_content: BigWig
Submission date Sep 14, 2021
Last update date Mar 25, 2022
Contact name KUNHUA QIN
Phone 2153603146
Organization name Children's Hospital of Philadelphia
Lab Gerd A Blobel
Street address 3615 Civic Center Blvd, 315H
City Philadelphia
State/province PA
ZIP/Postal code 19104
Country USA
Platform ID GPL30173
Series (2)
GSE180860 NFI factors repress fetal hemoglobin in adult erythorid cells [CUT&RUN]
GSE180871 NFI factors repress fetal hemoglobin in adult erythorid cells
BioSample SAMN21434703
SRA SRX12187919

Supplementary file Size Download File type/resource 77.1 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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