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Status |
Public on Nov 05, 2023 |
Title |
RNA-H9-WT-5i-2 |
Sample type |
SRA |
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Source name |
human embryo stem cell
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Organism |
Homo sapiens |
Characteristics |
cell line: H9 culture medium: 5i/L/A genotype: wild type rip antibody: none
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Growth protocol |
H9 hESCs were cultured using mTeSR medium (Stemcell technologies, Cambridge, USA). Cells were cultured on Matrigel (Fisher Scientific) -coated culture dishes, with daily medium change. H9 hESCs were passaged as small cluster using Versene solution (Thermo Fisher Scientific, USA). R1 mESCs were cultured on mouse embryonic fibroblast feeder cells inactivated with mitomycin-C (Sigma-Aldrich), using mouse ESC (mESC) medium (knockout DMEM, Life Technologies, Carlsbad, CA, USA) supplemented with 15% knockout serum replacer (Life Technologies, Carlsbad, CA, USA), 1×GlutaMAX (Life Technologies, Carlsbad, CA, USA), 1×Anti-Anti (Life Technologies, Carlsbad, CA, USA), 1% MEM non-essential amino acids (Life Technologies, Carlsbad, CA, USA), 0.1 mM 2-Mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA) and 1000 Units/ml ESGRO-mLIF (Millipore, Burlington, MA, USA), at 37˚C in 5% CO2. R1 mESCs were passaged as single cell using 0.05%-trypsin-EDTA (Life Technologies, Carlsbad, CA, USA). H9 cells were induced to the naïve state using the 5i method (Theunissen et al., 2014). Briefly, H9 cells maintained on Matrigel were seeded onto feeder cells for two generations for acclimation with mTeSR medium, and were dissociated into single cells using Accutase (Thermo Fisher, USA). Dissociated H9 cells (2 × 105, for one well of a 12-well plate) were cultured with mTeSR supplemented with 10 μM Y27632 (Stemcell technologies, Cambridge, USA) for one day before switching to the 5i medium (DMEM/F12 : Neural basal = 1:1 mix, 1×N, 1× B27, 1×GlutaMAX, 1% NEAA, 0.1 mM BME, 1 μM PD0325901, 3 μM Chir99021, 0.5 μM SB590885, 1 μM WH-4-023, 10 μM Y27632, 20 ng/mL hLIF, 20 ng/mL Activin A, 8 ng/mL FGF2). Induced naïve state colonies with translucent edges were visible after 10 days of culture, and passaged using Accutase (Thermo Fisher, USA).
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Extracted molecule |
total RNA |
Extraction protocol |
For RNA-seq, total RNA was extracted from the cells using TRIzol® Reagent (Invitrogen) and genomic DNA was removed using DNase I (TaKara). For RIP-sequencing, RNA was extracted from RNA precipitation preparations. Then RNA quality was determined using 2100 Bioanalyser (Agilent) and quantified using the ND-2000 (NanoDrop Technologies). High-quality RNA sample (OD260/280=1.8~2.2, OD260/230≥2.0, RIN≥6.5, 28S:18S≥1.0, >10μg) is used to construct sequencing library. For RIP-seq,RNA immunoprecipitation was performed with reversible cross-linking of the cells, as previously described (Niranjanakumari et al., 2002). In brief, 1×107 cells were washed three times with pre-chilled PBS and resuspended in 10 mL of PBS, cells were fixed with 1% formaldehyde (diluted from 37% HCHO/10% methanol stock solution, Sigma-Aldrich) at room temperature for 10 minutes with slow mixing. Crosslinking reactions was quenched by the addition of glycine (pH 7.0) to a final concentration of 0.25 M, followed by incubation at room temperature for 5 minutes. The cells were harvested by centrifugation and washed twice with ice-chilled PBS. Fixed cells were resuspended in 2 mL of RIPA buffer containing protease inhibitors (Roche) and RNAase inhibitors (Ambion), and sonication was used to solubilize RNPs. Insoluble material was removed by centrifugation at 16,000g for 10 minutes at 4 °C. The supernatant was mixed with the antibody and incubated overnight at 4˚C with rotation, followed by incubation with protein A/G beads. The beads were collected by centrifugation at 6000 rpm for 30s and washed five times with wash buffer (50 mM Tris–Cl, pH 7.5, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1 mM EDTA, 1 M NaCl and 0.2 mM PMSF). The beads containing the immunoprecipitated samples are collected and resuspended in resuspended buffer (50 mM Tris–Cl, pH 7.0, 5 mM EDTA, 10 mM DTT and 1% SDS). Samples (resuspended beads) are incubated at 70°C for 45 minutes to reverse the crosslinks. The RNA was extracted from these samples using Trizol reagent (Invitrogen) and used for RIP-Seq. RNA libraries were prepared for sequencing using standard Illumina protocols
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
The raw paired end reads were trimmed and quality controlled by Trimmomatic with parameters (SLIDINGWINDOW:4:15 MINLEN:75) (version 0.36) clean reads were separately aligned to reference genome with orientation mode using bowtie2 MACS2 for peak calling : The candidate peak region was extended to be long enough for modeling. Dynamic Possion Distribution was used to calculated p-value of the specific region based on the unique mapped reads. The region would be defined as a peak when p-value < le-03. For RNA-seq, clean reads were separately aligned to reference genome with orientation mode using hisat2.The quality assessment of these data were taken by qualimap_v2.2.1.Use htseq to count each gene reads. Genome_build: mm10 for R1 cell line Genome_build: hg38 for H9 cell line Supplementary_files_format_and_content: bdg and bw files were used for represent RIP-seq data Supplementary_files_format_and_content: xlsx file with raw gene counts for every gene and every sample
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Submission date |
Feb 28, 2022 |
Last update date |
Nov 05, 2023 |
Contact name |
Gufa Lin |
E-mail(s) |
lingufa@tongji.edu.cn
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Organization name |
Tongji University
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Department |
Key Laboratory of Spine and Spinal Cord Injury Repair and Regeneration of Ministry of Education, Orthopaedic Department of Tongji Hospital, School of Life Sciences and Technology, Tongji University
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Street address |
1239 Siping road Tongji university
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City |
Shanghai |
ZIP/Postal code |
200092 |
Country |
China |
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Platform ID |
GPL24676 |
Series (1) |
GSE197608 |
Short C-terminal Musashi-1 proteins regulate pluripotency states in embryonic stem cells |
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Relations |
BioSample |
SAMN26209681 |
SRA |
SRX14271988 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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