 |
 |
GEO help: Mouse over screen elements for information. |
|
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 Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
 |