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Sample GSM7676708 Query DataSets for GSM7676708
Status Public on Mar 19, 2024
Title HKC_sgVHL_sg1_MeRIP_Rep1
Sample type SRA
 
Source name HKC
Organism Homo sapiens
Characteristics cell line: HKC
treatment: sgVHL_sg1
Treatment protocol METTL3 and IGF2BP1 lentiviral shRNA constructs were purchased from Sigma-Aldrich. VHL, PIK3R3, HIF1a, HIF2a, ARNT, IGF2BP2, IGF2BP3, p85a and p85b sgRNAs were cloned into pLentiCRISPR V2 -Puromycin vector. siRNAs were purchased from Invitrogen. Target sequences of shRNA, sgRNA and siRNA are listed in supplementary tables in the manuscript.
Growth protocol HKC cells are obtained from Dr. Kimryn Rathmell from Vanderbilt University. All the cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, 11995073) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. Mycoplasma testing was routinely carried out with MycoAlert® PLUS Mycoplasma Detection Kit (Lonza, LT07-703) to ensure cells are mycoplasma free.
Extracted molecule total RNA
Extraction protocol Cellular RNA was extracted using the RNeasy mini kit (Qiagen, 74104) according to the manufacturer’s instructions. Complementary DNA (cDNA) was synthesized iScript Reverse Transcription Supermix (Bio-Rad, 1708841). RIP was performed using a modified native RNA immunoprecipitation procedure as previously described. In briefly, approximately 3x10^7 HKC cells were lysed with lysis buffer (100mM KCl, 5mM MgCl2, 10mM HEPES pH 7.0, 0.5% NP-40, 1mM DTT, supplemented with RNase inhibitor and EDTA-free Protease inhibitor before using). Incubate the cell lysate with protein-A/G magnetic beads that immobilized with corresponded antibodies for overnight at 4 °C. Wash the beads 6 times with NT-2 buffer (50mM Tirsh-HCl pH 7.4, 250mM NaCl, 1 mM MgCl2, 0.05% NP-40, 20mM EDTA, 1mM DTT, supplemented with RNase inhibitor before using). Input and co-immunoprecipitated RNAs were first digested with Proteinase K buffer (NT-2 buffer supplemented with 1% SDS and 1.2 mg/ml Proteinase K) at 55 °C for 30 min and then recovered by TRIzol according to the supplier’s instructions.
Immunoprecipitated RNA was extracted with RNeasy mini kit (Qiagen, 74106). cDNA library was prepared with NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (NEB, E7775). Samples were subjected for qPCR analysis or sequenced on the Illumina NextSeq 500 NGS system as 75 bp pair-ended reads.
 
Library strategy RIP-Seq
Library source transcriptomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description MeRIP-seq
Data processing The MeRIP and input library reads were aligned to aligned to mycoplasma genome to assess contamination followed by alignment to human reference genome (hg19) with STAR. MarkDuplicates (Picard) was used for identifying and removing duplicates reads. DESeq2 was used to call the differential expression genes (DEG) between Ctrlsg and VHL knockout groups. The m6A peaks were called by MeTDiffpackage with default parameter by combining signals from all the biological triplicates. Homer was used to search for the enriched motif in the m6A peaks, and the m6A peak distribution across the whole transcriptome was called using the Guitar package. A convolutional neural network model (MATK, http://matk.renlab.org/#/home) was used identify the specific m6A site from the detected peaks. Pathway enrichment analysis were performed using Enrichr (https://maayanlab.cloud/Enrichr/enrich).
Assembly: hg19
Supplementary files format and content: m6A bigwig files contain read-depth normalized per-base signal for each m6A sample and replicate.
Supplementary files format and content: Excel files labeled *_MATK.xlsx contain m6A peaks across treatments as determined by MATK.
Supplementary files format and content: Excel files labeled *m6A_peaks_MeTDiff.xlsx contain m6A peaks per treatment as determined by MeTDiff.
Supplementary files format and content: Excel files labeled *diff_peaks_MeTDiff.xlsx contain differential m6A peaks between labeled treatments as determined by MeTDiff. Files are labeled as they were run in MeTDiff: treatment_vs_control.
Supplementary files format and content: Excel files labeled *DESeq2_feature_counts.xlsx contain per-gene counts for each treatment and replicate as determined by DESeq2.
Supplementary files format and content: Excel files labeled *DESeq2_diff_genes.xlsx contain differentially expressed genes between labeled treatments as determined by DESeq2. Files are labeled as they were run in DESeq2: treatment_vs_control. File contains DESeq2 output for both sgVHL_sg1 vs sgCtrl and sgVHL_sg2 vs sgCtrl
 
Submission date Aug 02, 2023
Last update date Mar 19, 2024
Contact name Austin J Hepperla
E-mail(s) hepperla@unc.edu
Organization name University of North Carolina at Chapel Hill
Department Genetics
Street address 7018B Mary Ellen Jones Building
City Chapel Hill
State/province NC
ZIP/Postal code 27599
Country USA
 
Platform ID GPL18573
Series (1)
GSE239892 Von Hippel Lindau tumor suppressor controls m6A-dependent gene expression in renal tumorigenesis
Relations
BioSample SAMN36812152
SRA SRX21229198

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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