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| Status |
Public on Mar 23, 2022 |
| Title |
CUTRUN_NFIA_Primary_Perpheral Blood_Invitrogen#535936 |
| Sample type |
SRA |
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| Source name |
Primary_Peripheral Blood
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| Organism |
Homo sapiens |
| Characteristics |
differentiation: D11
antibody: NFIA
biological replicate: NA
treatment: no
tissue: Peripheral Blood
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| 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
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| 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.
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| 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 (dx.doi.org/10.17504/protocols.io.bcuhiwt6) 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 (dx.doi.org/10.17504/protocols.io.wvgfe3w) 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.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Data processing |
Library strategy: CUT&RUN-seq
CUT&RUN sequencing reads were analyzed by CUT-RUNTOOLS-2.0 (https://github.com/fl-yu/CUT-RUNTools-2.0).
Genome_build: hg38
Supplementary_files_format_and_content: BigWig
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| Submission date |
Sep 14, 2021 |
| Last update date |
Mar 25, 2022 |
| Contact name |
KUNHUA QIN |
| E-mail(s) |
qink@chop.edu, kunhua.qin@gmail.com
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| Phone |
2153603146
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| Organization name |
Children's Hospital of Philadelphia
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| Lab |
Gerd A Blobel
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| Street address |
3615 Civic Center Blvd, 315H
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| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
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| 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 |
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| Relations |
| BioSample |
SAMN21434703 |
| SRA |
SRX12187919 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5578008_CUTRUN_NFIA_Primary_Perpheral_Blood_Invitrogen_535936.cpm.norm.bw |
77.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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