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| Status |
Public on Feb 18, 2022 |
| Title |
HOTTIP-KO-NG-Capture-C-seq |
| Sample type |
SRA |
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| Source name |
MOLM13 leukemia cells
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| Organism |
Homo sapiens |
| Characteristics |
tissue: peripheral blood
cell type: Human-derived acute myeloid leukemia cells
cell line: MOLM13 leukemia cells
gene fusion: Gene fusion KMT2A-MLLT3 (MLL-MLLT3; MLL-AF9)
genotype: HOTTIP-KO
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| Treatment protocol |
Immediately extract RNA from LK, LSK cells and MOLM13 AML cells without any treatment
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| Growth protocol |
Total bone marrow cells were isolate from WT and Hottip-Tg mice, and sorted with Lin-, Sca1+ and Kit+ markers. Human MOLM13 leukemia cells were culture at RMPI1640 medium with 10% FBS media.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA samples extracted with Trizon and treated with Dnase I. ChIP Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with CTCF, SA1, SA2, RAD21, H3K4me3 and H3K27me3 antibody (antibody: Anti-CTCF, Cell Signaling Technology, Cat# 2899; Anti-SA1, Abcam, Cat# ab4457; Anti-SA2, Abcam, Cat# ab4464; Anti-RAD21, Abcam, Cat# ab992; Anti-H3K4me3, Millipore, Cat No. 04-745; Anti-H3K27me3, Millipore, Cat No. 07-449). ATAC-seq samples derived according to Nextera Tn5 Transposase kits. DNA:RNA immunoprecipitation sequencing (DRIP-seq) was performed as described previously with s9.6 antibody (Anti-DNA-RNA Hybrid [S9.6] Antibody, Kerafast, Cat# ENH001). NG-Capture-C assay was performed as previously described. Hi-C assay was performed to generate a genome-wide interaction as described previously with Arima-HiC Kit (Cat: A410030) (https://arimagenomics.com/) with minor modifications. Single cells were generated using Chromium Controller (10x Genomics), and scRNA-seq and scATAC-seq libraries were constructed using chromium single cell 3’ reagent kits v2 (10x Genomics, California USA) according to the manufacturer’s recommendations.
RNA-seq libraries were prepared according to TruSeq Stranded mRNA Library Prep (#20020594). ChIP-seq libraries were prepared according to Illumina's instructions accompanying the TruSeq ChIP Library Preparation Kit (#IP-202-1012). ATAC-seq libraries were prepared according to Nextera DNA Library Prep Kit (#FC-121-1030). NG-Capture-C-seq libraries were prepared according to using the Herculase II PCR kit (Agilent). . Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
single cell RNA-seq, ATAC-seq, ChIP-seq, DRIP-seq, DRIPc-seq, NG-Capture-C-seq and HiC-seq
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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: Capture-C-seq
Standard Illumina software base-calling and quality-control filtering was applied Sequences (Paired end)
Paired end HiC-seq and scRNA-seq and scATAC-seq reads from mice were aligned to the mm9 genome assembly using BWA, and NG capture-C-seq, ChIP-seq, DRIP-seq and DRIPc-seq reads from human cells were aligned to the hg19 genome assembly using BOWTIE2 default parameters.
Peak calling was performed using MACS algorithm (version 2.1.1)
Genome coverage tracks were created using deepTools version 3.0
Genome_build: mm9, hg19
Supplementary_files_format_and_content: tab-delimited text files include FPKM values for scRNA-seq and scATAC-seq Samples and peaks for DRIP-seq , DRIPc-seq, ChIP-seq, NG-capture-C and HIC-seq.
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| Submission date |
Jan 18, 2021 |
| Last update date |
Feb 18, 2022 |
| Contact name |
Suming Huang |
| E-mail(s) |
huanglabseq@hotmail.com
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| Organization name |
Penn State University
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| Department |
Pediatrics
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| Street address |
500 University Dr.
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| City |
Hershey |
| State/province |
PA |
| ZIP/Postal code |
17033 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE165049 |
HOTTIP reinforces CTCF-defined TAD boundaries by forming R-loops to drive Wnt/b-catenin target gene expression in AML leukemogenesis |
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| Relations |
| BioSample |
SAMN17376219 |
| SRA |
SRX9891948 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5025285_captureC_HOTTIP-peaks.bed.gz |
16.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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