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Sample GSM5923623

Query DataSets for GSM5923623

Status

Public on Nov 05, 2023

Title

RNA_seq-R1_WT_r1

Sample type

SRA

Source name

mouse embryo stem cell

Organism

Mus musculus

Characteristics

cell line: R1
culture medium: MES+LIF
genotype: wild type
rip antibody: none

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).

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

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

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

Submission date

Feb 28, 2022

Last update date

Nov 05, 2023

Contact name

Gufa Lin

E-mail(s)

lingufa@tongji.edu.cn

Organization name

Tongji University

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

Street address

1239 Siping road Tongji university

City

Shanghai

ZIP/Postal code

200092

Country

China

Platform ID

GPL24247

Series (1)

GSE197608