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Sample GSM7720917 Query DataSets for GSM7720917
Status Public on Jul 24, 2024
Title K562, WT, caRNA, m5C-IP, rep2
Sample type SRA
 
Source name bone marrow
Organism Homo sapiens
Characteristics tissue: bone marrow
cell line: K562
cell type: lymphoblast cell
genotype: wildtype
antibody: m5C (Diagenode, #MAb-081-100)
Treatment protocol N/A
Growth protocol WT K-562 cells were kept in RPMI-1640 (Gibco, 61870036) with 10% fetal bovine serum (FBS, Gibco) and 1 μg/mL puromycin (Gibco, A1113803) at 37 °C and 5% CO2. mESCs were kept in DMEM (Gibco, 11995065) supplemented with 15% Stem Cell Qualified Fetal Bovine Serum, Heat Inactivated (Gemini Bio Products, 100-525), 1 × L-glutamine (Gibco, 25030081), NEAA (Gibco, 25030081), LIF (MilliporeSigma, ESG1107), 1 × β-mercaptoethanol (Gibco, 21985023), 3 μM CHIR99021 (STEMCELL Technologies, 72052) and 1 μM PD0325901 (STEMCELL Technologies, 72182) at 37 °C and 5% CO2. The medium was replaced every day. ES cells were passaged on gelatin-coated plates twice to clear feeder cells before experiments.
Extracted molecule total RNA
Extraction protocol Total RNAs from whole cell or the chromatin-associated fractions were randomly fragmented by incubation at 94 °C for 4 minutes using 1× fragmentation buffer (NEB E6186A). Fragmentation was stopped by adding 1× Stop Solution. Spike-in RNAs were added to each sample. Four microgram anti-m5C antibody (Diagenode, MAb-081-100) was conjugated with 30 μL of protein G beads (Invitrogen, 1003D) in 300 μL IP buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.05% Triton X-100 (v/v)) for two hours at 4 °C on a rotating wheel. The same procedure was performed for a control reaction using mouse IgGs (Abcam ab37355). Bead-antibody complexes were washed three times with IP buffer and finally brought to 250 μL with IP buffer. After heat denaturation and quick chill on ice, 10-μg samples of RNA were added to the bead-antibody complexes and incubated with 1 μL SUPERase•In™ RNase Inhibitor (Invitrogen, AM2694) overnight at 4 °C on a rotating wheel. After several washes with IP buffer, RNA was incubated in 100 μL elution buffer (5 mM Tris-HCl pH 7.5, 1 mM EDTA, 0.05% SDS, and 200 μg Proteinase K (Invitrogen 25530049)) for 1 hour at 50 °C. Beads were removed by centrifugation in a microcentrifuge, and the supernatant was purified with RCC-5 without size selection.
Libraries were constructed with SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa Bio, 634411) according to the manufacturer’s instructions.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Data processing Raw reads were trimmed with Trimmomatic (version 0.39), then aligned to mouse (mm10) or human (hg38) genome and transcriptome using HISAT2 (version 2.1.0).
Annotation files (version M19 for mouse, and version v29 for human in gtf format) were downloaded from GENCODE database (https://www.gencodegenes.org/).
Mapped reads were separated by strands with samtools (version 1.16.1) and m5C peaks on each strand were called using MACS2 (version 2) with parameter ‘--nomodel, --keep-dup all, -g 7e8, -extsize 150’ separately. In this regard, the genome size was estimated based on reads coverage obtained from input samples. Significant peaks with q < 0.01 identified by MACS2 were considered.
Assembly: mm10 for mouse and hg38 for human
Supplementary files format and content: raw counts for each Sample, peaks in mESCs (except for input samples)
Library strategy: MeRIP-Seq
 
Submission date Aug 21, 2023
Last update date Jul 24, 2024
Contact name Xiaoyang Dou
E-mail(s) xiaoyang.dou@sibcb.ac.cn
Organization name Center for Excellence in Molecular Cell Science, CAS
Street address 320 Yue Yang Road
City Shanghai
ZIP/Postal code 200031
Country China
 
Platform ID GPL24676
Series (2)
GSE241308 TET2 regulates histone H2AK119ub and leukemogenesis through LTR RNA m5C oxidation [seq_RNA-caRNA-m5C-MeRIP]
GSE241347 Chromatin-associated LTR RNA m5C oxidation by TET2 regulates leukemogenesis
Relations
BioSample SAMN37096166
SRA SRX21436568

Supplementary file Size Download File type/resource
GSM7720917_K562_WT_caRNA_m5C-IP_rep2_counts.txt.gz 203.3 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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