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| Status |
Public on Apr 22, 2024 |
| Title |
KYSE180 cells, shEZH2#1_3 |
| Sample type |
SRA |
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| Source name |
KYSE180 cell
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| Organism |
Homo sapiens |
| Characteristics |
cell line: KYSE180 cell
cell type: esophageal squamous cell carcinoma
genotype: EZH2 knockout
treatment: 6 Gy IR
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| Treatment protocol |
All cells were exposed 6 Gy IR
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| Growth protocol |
All cells were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) at 37˚C in a 5% CO2 atmosphere
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's procedure. The RNA amount and purity of each sample was quantified using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA). The RNA integrity was assessed by Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Poly (A) RNA is purified from 1μg total RNA using Dynabeads Oligo (dT)25-61005 (Thermo Fisher, CA, USA) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using Magnesium RNA Fragmentation Module (NEB, cat.e6150, USA) under 94℃ 5-7min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA by SuperScript™ II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesise U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA）. An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR by the following conditions: initial denaturation at 95℃ for 3 min; 8 cycles of denaturation at 98℃ for 15 sec, annealing at 60℃ for 15 sec, and extension at 72℃ for 30 sec; and then final extension at 72℃ for 5 min. The average insert size for the final cDNA library was 300±50 bp.
RNA libraries for RNA-seq were prepared using TruSeq Stranded mRNA Library Prep Kit following manufacture's
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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 |
We performed the 2×150bp paired-end sequencing (PE150) on an illumina Novaseq™ 6000 (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.
Fastp software (https://github.com/OpenGene/fastp) were used to remove the reads that contained adaptor contamination, low quality bases and undetermined bases with default parameter.
Then sequence quality was also verified using fastp.
We used HISAT2 (https://ccb.jhu.edu/software/hisat2) to map reads to the reference genome of Homo sapiens GRCh38
The mapped reads of each sample were assembled using StringTie (https://ccb.jhu.edu/software/stringtie) with default parameters
Then, all transcriptomes from all samples were merged to reconstruct a comprehensive transcriptome using gffcompare (https://github.com/gpertea/gffcompare/).
After the final transcriptome was generated, StringTie and was used to estimate the expression levels of all transcripts.
StringTie was used to perform expression level for mRNAs by calculating FPKM (FPKM = [total_exon_fragments / mapped_reads(millions) × exon_length(kB)]).
Assembly: hg38
Supplementary files format and content: tab-delimited text file includes raw counts for each Sample
Supplementary files format and content: tab-delimited text files include RPKM values for each Sample
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| Submission date |
Apr 16, 2024 |
| Last update date |
Apr 22, 2024 |
| Contact name |
嘉昊 管 |
| E-mail(s) |
J_HGuan@126.com
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| Phone |
18755271875
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| Organization name |
安徽医科大学第一附属医院
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| Street address |
安徽省蚌埠市龙子湖区凤阳东路536号4幢1单元1002
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| City |
蚌埠市 |
| State/province |
安徽省 |
| ZIP/Postal code |
233000 |
| Country |
China |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE264079 |
The effect of EZH2 knockdown on gene expression in KDM6A-deficient KYSE180 cells following exposure to ionizing radiation [RNA-seq] |
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| Relations |
| BioSample |
SAMN40986301 |
| SRA |
SRX24277135 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8209870_shEZH2_1_3.txt.gz |
187.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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