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| Status |
Public on Mar 11, 2024 |
| Title |
Naive-H9_RNAseq_rep1 |
| Sample type |
SRA |
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| Source name |
H9
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| Organism |
Homo sapiens |
| Characteristics |
cell line: H9
cell type: ESC
genotype: WT
treatment: navie induced medium
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| Treatment protocol |
The medium was changed every other day. The dome-shaped colonies were dissociated with Accutase (Thermo Fisher Scientific, catalog. no. A1110501) into single cells, and then passaged onto new Matrigel- or feeder-coated plates every 3 - 4 days.
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| Growth protocol |
The naive hESCs (also named as prEpiSCs) were derived from the LTR7-GFP hESC_H9 cell line. The basal culture medium (500 ml) is prepared as follows, 240 ml DMEM/F12 (Thermo Fisher Scientific, catalog. no. 21331020), 240 ml Neurobasal medium (Thermo Fisher Scientific, catalog. no. 21103049), 2 mM L-glutamine (Thermo Fisher Scientific, catalog. no. 25030081), non-essential amino acids (Thermo Fisher Scientific, catalog. no. 11140050), 1% N2 supplement (Thermo Fisher Scientific, catalog. no. 17502048), 2% B27 supplement (Thermo Fisher Scientific, catalog. no. 12587010, minus vitamin A), 100 mg/ml Vitamin C (Sigma, catalog no. A4403), 50 ng/ml Bovine Albumin Fraction V (Thermo Scientific, cat. no. 15260-037), 20 ng/ml IL6 (PeproTech, catalog. no. AF-200-06), 20 ng/ml sIL-6R (PeproTech, catalog. no. 200-06R), 0.1 mM β-mercaptoethanol (Thermo Fisher Scientific, catalog. no. 21985023), Primocin (InvivoGen, catalog. no. ant-pm-2). The naive/prEpiSCs were cultured in the advanced GIX medium (basal culture medium plus chemical cocktail [0.2 μM Trametinib (GSK112022, Selleck S2673), 0.3 μM PD0325901, 2.0 μM XAV939, 0.5 μM Go6983]).IL6, IL-6R, Trametinib, PD0325901, XAV939, Go6983
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| Extracted molecule |
total RNA |
| Extraction protocol |
the sample used to perform RNA-seq are prepared based on the commonly used method.
AS for RNA-seq, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in NEB Next First Strand Synthesis Reaction Buffer (5x). First strand cDNA was synthesized using random hexamer primer and M-MLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3’ ends of DNA fragments, NEB Next Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, USA). Then 3 μl USER Enzyme (NEB, USA) was used with size-selected and cDNA adaptor ligated at 37 °C for 15 min followed by 5 min at 95 °C prior to PCR. PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index Primer. At last, PCR products were purified using AMPure XP system and library quality assessed on the Agilent Bioanalyzer 2100 system. Cluster of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit (Illumina) according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina Hiseq platform.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 1500 |
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| Data processing |
The adapter sequences of RNA-seq data and low-quality reads with Phred score <5 were deleted with Cutadapt before further processing.
Clean reads of RNA-seq were mapped to hg38 using HISAT2(2.1.0)
Expression levels of all genes were count.txted and expression profile matrix was constructed with featureCounts (2.0.4)
Differential expression analysis was calculated in R using DESeq2(1.18.1), with padj < 0.05 and fold change > 2 or < 1/2
Assembly: hg38
Supplementary files format and content: gene FPKM text file
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| Submission date |
Mar 06, 2024 |
| Last update date |
Mar 11, 2024 |
| Contact name |
Niannian Li |
| Organization name |
Nankai University
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| Department |
college of Life Sciences
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| Street address |
No. 94 Weijin Road, Nankai District, Tianjin
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| City |
Tianjin |
| State/province |
Tianjin |
| ZIP/Postal code |
300071 |
| Country |
China |
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| Platform ID |
GPL18460 |
| Series (2) |
| GSE260994 |
Single-cell 3D genome structure reveals distinct human pluripotent states (RNA-Seq) |
| GSE260995 |
Single-cell 3D genome structure reveals distinct human pluripotent states |
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| Relations |
| BioSample |
SAMN40286084 |
| SRA |
SRX23855201 |
| 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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