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| Status |
Public on Jan 03, 2024 |
| Title |
shTDP43_3 |
| 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
genotype: TDP-43 knockdown
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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 |
hMe-Seal was performed as previously described[2]. About 2 millions of cells were lysed in lysis buffer (100 mM Tris-HCl pH 8.0, 5 mM EDTA, 200 mM NaCl, 0.2% SDS) with addition of proteinase K at 55℃ overnight. gDNA was then extracted by phenol-chloroform extraction followed by ethanol precipitation. 5 μg of gDNA was sonicated into small fragments (~100-500 bp) using Covaris Focused-Ultrasonicator Me220. Fragmented gDNA was treated with β-glucosyltransferase (β-GT, New England BioLabs) in the presence of UDP-6-N3-Glu (Jena Bioscience) to selectively label 5hmC into N3-5-gmC. After AMPure XP bead (Beckman Coulter) purification, N3-5-gmC was further labeled with disulfide DBCO biotin (Click Chemistry Tools) to install biotin. DNA was purified with AMPure XP bead and pulled down with 50 μL of Dynabeads MyOne Streptavidin C1 beads (Invitrogen). 5hmC-fragments were released from the beads by adding Dithiothreitol (DTT), purified with AMPure XP bead and quantified by Qubit. 5 ng of 5hmC-DNA was 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 |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| 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)
Library strategy: 5hmC-Seal-seq
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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) |
| GSE230405 |
TDP-43 stable deficiency leads to transcriptional dysregulation and transposable elements activation via global R-loops and 5hmC landscape alteration [5hmC-Seal-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 |
SAMN34353156 |
| SRA |
SRX20083067 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7221624_shTDP43_3_5hmC_peaks.narrowPeak.gz |
3.0 Mb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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