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| Status |
Public on Jan 03, 2024 |
| Title |
DRIP_shNC_2 |
| Sample type |
SRA |
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| Source name |
SH-SY5Y
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| Organism |
Homo sapiens |
| Characteristics |
cell line: SH-SY5Y
cell type: Neuroblastoma
chip antibody: S9.6
genotype: control
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| Treatment protocol |
shRNA oligos (shNC and shTDP-43) synthesized from Integrated DNA Technologies (IDT) were annealed and inserted into linearized lentiviral vector pLVX-U6-shRNA1 (Genprice, #PVT2302) using BamH I and EcoR I. The shRNA constructs were then used for lentivirus production. 293T packaging cells were seeded at 5x106 cells per 10 cm tissue culture plate the day before transfection. 1.3 pmol of psPAX2 (Addgene, #12260), 0.72 pmol of pMD2.G (Addgene, #12259), and 1.64 pmol of lentiviral plasmid were co-transfected into 293T cells using polyethylenimine MW 25000 (PEI 25K, Polyscience, #23966-100) at a ratio of 1:3 (w:w). Virus-containing medium was harvested 48 and 72 hr post-transfection and passed through a 0.45 μm PES filter (VWR, #76211-008). The virus was then concentrated 100-fold with Lenti-X concentrator (TaKaRa, #631231). Virus infection was performed by adding 20 μL of lentivirus to ~800,000 SH-SY5Y cells (MOI: 1:3) in a 6-well plate. TDP-43 stable knockdown cells were selected by Puromycin at a concentration of 2 μg/mL for 2 weeks and maintained in medium with 1 μg/mL of Puromycin for long-term use.
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| Growth protocol |
Human neuroblastoma cells SH-SY5Y (ATCC) were purchased from ATCC and cultured in Eagle's Minimum Essential Medium (EMEM) (Corning) and Ham’s F12 medium (Corning) (1:1, v:v) supplemented with 15% v/v fetal bovine serum (FBS) at 37℃ and 5% carbon dioxide (CO2).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, for each replicate, about 2-3 million cells were harvested for nucleic acid isolation. Nucleic acids were extracted from the cells by SDS/proteinase K treatment followed by phenol-chloroform extraction using MaXtract High Density Tubes (Qiagen) and ethanol precipitation. Nucleic acids were fragmented with a group of restriction enzymes (BsrGI, EcoRI, HindIII, SspI, XbaI, 30 unit each). Digested nucleic acids were recovered by phenol-chloroform extraction. 6 μg of fragmented nucleic acids were treated with RNase H for 6 hr as negative control. 6 μg of digested DNA and RNase H-treated DNA were immunoprecipitated with 8 μL of DNA-RNA structure-specific S9.6 antibody (Millipore) overnight at 4℃. 40 ul of protein G beads (Invitrogen) were used to pull down the DNA:RNA hybrid at 4℃ for 2 hr. The beads were washed twice. DNA-RNA hybrids were eluted in 0.5% SDS and cleaned up by phenol-chloroform extraction. Immunoprecipitated DNA-RNA hybrids were quantified by Qubit (Thermo Fisher Scientific). 10 ng of DNA was then subjected to library construction.
For DRIP-seq, immunoprecipitated DNA was first sonicated into small fragments (~100-500 bp) using Covaris Focused-Ultrasonicator Me220. Fragmented ends of DRIPed-DNA and 5hmC-DNA were blunted, 5′ phosphorylated and 3’ A-tailed by NEBNext Ultra II End Prep Enzyme Mix following the manufacturer’s instruction (New England Biolabs, E7645L). Adaptor was ligated to the DNA with optimized working concentration and USER enzyme was used for U excision to yield adaptor-ligated double-strand DNA followed by cleanup and size selection with AMPure XP bead. The adaptor-ligated DNA was PCR-amplified using barcoded PCR primers (New England Biolabs, E7335L, E7500L). After AMPure XP bead purification, Qubit quantification, the size and quality of the libraries was assessed by 2100 Bioanalyzer system. Libraries were sent to Admera Health for sequencing with Illumina.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
DRIP-seq, hMe-Seal, ATAC-seq reads were first aligned to human genome sequence (hg38) by Bowtie 2 version 2.4.4 with default parameter.
Aligned reads in bam file were sorted by genomic coordinate by samtools.
Peaks for DRIP-seq and hMe-Seal libraries were identified by MACS2 version 2.2.6 with their corresponding input bam file
Assembly: hg38
Supplementary files format and content: narrowPeak (except for Input sample)
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| Submission date |
Apr 24, 2023 |
| Last update date |
Jan 03, 2024 |
| Contact name |
Bing Yao |
| E-mail(s) |
bing.yao@emory.edu
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| Phone |
3523596473
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| Organization name |
Emory University
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| Department |
Department of Human Genetics
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| Street address |
615 Michael Street
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| City |
Atlanta |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE230407 |
TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global R-loops and 5hmC landscape alteration [DRIP-Seq] |
| GSE230410 |
TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global R-loops and 5hmC landscape alteration |
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| Relations |
| BioSample |
SAMN34353175 |
| SRA |
SRX20083260 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7221633_DRIP_shNC_2_peaks.narrowPeak.gz |
1.4 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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