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| Status |
Public on Sep 06, 2024 |
| Title |
scNanoSeq-CUT&Tag_K562_drug_treatment_DMSO_H3K27ac_Abcam_ab4729 |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562
cell type: Lymphoblast
chip antibody: H3K27ac (Abcam,#ab4729)
treatment: DMSO
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| Treatment protocol |
The K562 cells were treated with 5 μM 5-azacytidine (5-AZA, Sigma, cat. no. A2385) for 5 days and the medium was exchanged every 2 days. For the control group, DMSO with the same volume of 5-azacytidine was added to the medium. The collected cells were washed and the clean cells were for scNanoSeq-CUT&Tag experiment. As for isolation of mouse blood cells, the 3 ml of whole blood from 8-week-old male B6D2F1 was firstly mixed with 30 ml of 1x red blood cell lysis buffer, incubated at room temperature for 15 min, and centrifuged at 400 x g for 20 min to remove red blood cells. And then the cell pellets were washed twice with 10ml of PBS and were stained with the following antibodies: APC-Cy7 anti-mouse CD45 (Biolegend, 103115), PE anti-mouse CD3 (Biolegend, 100205), CD19 (Biolegend, 115537), FITC anti-mouse CD4 (Biolegend, 100405), APC anti-mouse CD8a (Biolegend, 100711). After that the free antibodies were washed away, the cells were filtered with a 40 μm filter and resuspended with 500 ul of PBS buffer containing 0.5% BSA for subsequent cell sorting. Finally, CD45+ immune cells, CD45+CD3+CD19-CD4+ T cells, CD45+CD3+CD19-CD8+ T cells, CD45+CD3-CD19+ B cells, CD45+CD3-CD19- monocytes were collected for subsequent scNanoSeq-CUT&Tag experiment. As for isolation for testicular cells, the testes from 8-week-old male B6D2F1 mouse were collected and dissociated into single cell suspension with 0.5mg/ml of collagenase type I, 0.25% of trypsin and 5mg/ml of DNase I. After that the digestion was stopped by adding 0.5 ml of FBS, the cells were filtered through a 70 μm filter and were washed twice with PBS. Finally, the clean testicular cells were used for scNanoSeq-CUT&Tag experiment.
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| Growth protocol |
The human chronic myelogenous leukemia (CML) cell line K562 was grown in RPMI1640 medium (Gibco; 11875093) supplemented with 15% fetal bovine serum (FBS), 1% penicillin-streptomycin (Gibco, 15140122) and 2mM GlutaMAX (Gibco, 35050061). The human embryonic kidney cell line HEK293T and the mouse embryonic fibroblast cell line 3T3 were both grown in DMEM medium (Gibco, 11995040) supplemented with 10% FBS and 1% penicillin-streptomycin. The two human B-lymphocyte cell lines GM12878 and HG002 (NA24385) were both grown in RPMI1640 medium supplemented with 15% FBS and 1% penicillin-streptomycin. The human T-lymphocyte cell line H9 was grown in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin. The human foreskin fibroblast cell line HFF1 was grown in DMEM medium supplemented with 15% FBS and 1% penicillin-streptomycin. All cell lines were validated to be free of mycoplasma contamination (Yeasen, 40612ES25).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Firstly, the scCUT&Tag was performed as pervious described with some modifications. Approximately 50,000 to 300,000 cells were collected, washed and incubated with specific antibodies against chromatin modifications or transcription factors. After tagmentation by pG-Tn5, single cells were sorted into 96-well plate containing cell lysis buffer with 24bp cell barcodes in each well and processed to preamplification to obtain long-read amplicons. And then the PCR product of 48 cells with different cell barcodes was pooled, purified and processed to further PCR amplification with sample barcodes introduced during amplification. Finally, the purified DNA with different barcodes can be pooled together for library construction.
Approximately 1ug of purified DNA was diluted to a volume of 48 μl for library construction by using SQK-LSK110 Ligation Sequencing kit according to the manufacturer's protocol.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
PromethION |
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| Description |
scNanoSeq-CUT&Tag of K562_drug_treatment_DMSO (H3K27ac)
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| Data processing |
Base calling was performed to convert the electrical signals into raw fastq files using Guppy (v6.5.7). The raw fastq data were demultiplexed into single cell files according to the single-cell barcodes sequence using nanoplexer (v0.1). Two successive demultiplexing processes were performed on the outer barcodes and inner barcodes.Reads with low quality (q<7) and short length (<100 bp) were filtered using NanoFilt (v2.8.0). Adaptors were trimmed using cutadapt (v3.5). The clean reads were next mapped to reference genome of human (hg38) and mouse (mm10) using minimap2 (v2.26-r1175). Reads with low mapping quality (mapQ<30) and not primary alignment were filtered using samtools (v1.3). Then the aligned reads were sorted by coordinate and PCR duplicates were removed using samtools. To obtain chromatin modification signals, the aligned bam files were converted to bed format using the 'bamtobed' command of bedtools (v2.30.0). Then, we extracted the two ends of the long reads, centring on each end and extending 50bp on both sides respectively. The produced bed files recorded the chromatin modification signals for each single cell. The fragments file was read into ArchR (v1.0.1) for analysis. Peak calling was performed using SEACR (v1.3).
Assembly: hg38 for human; mm10 for mouse
Supplementary files format and content: Bigwig files recorded scNanoSeq-CUT&Tag signal strength tracks. Bed files recorded coordinates of scNanoSeq-CUT&Tag peaks. bedgraph.gz files recorded scNanoSeq-CUT&Tag signal strength tracks preduced in this expreiment.
Library strategy: scNanoSeq-CUT&Tag
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| Submission date |
Dec 09, 2023 |
| Last update date |
Sep 06, 2024 |
| Contact name |
Yuqing Guo |
| E-mail(s) |
[email protected]
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| Organization name |
Peking University
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| Department |
Biomedical Pioneering Innovation Center
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| Street address |
Peking University, No.5 Yiheyuan Road, Haidian District, Beijing, China
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| City |
BeiJing |
| State/province |
BeiJing |
| ZIP/Postal code |
100871 |
| Country |
China |
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| Platform ID |
GPL26167 |
| Series (1) |
| GSE249799 |
scNanoSeq-CUT&Tag: a long-read single-cell CUT&Tag sequencing method for efficient chromatin modification profiling within individual cells |
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| Relations |
| BioSample |
SAMN38339308 |
| SRA |
SRX22760389 |
| SRA |
SRX22760390 |
| SRA |
SRX22760393 |
| SRA |
SRX22760394 |
| SRA |
SRX22760397 |
| SRA |
SRX22760398 |
| SRA |
SRX22760406 |
| SRA |
SRX22760407 |
| SRA |
SRX22760411 |
| SRA |
SRX22760412 |
| SRA |
SRX22760417 |
| SRA |
SRX22760418 |
| SRA |
SRX22760424 |
| SRA |
SRX22760425 |
| SRA |
SRX22760426 |
| SRA |
SRX22760427 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7964500_K562_H3K27ac_DMSO.bed.gz |
879.7 Kb |
(ftp)(http) |
BED |
| GSM7964500_K562_H3K27ac_DMSO.bw |
42.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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