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| Status |
Public on Jun 01, 2020 |
| Title |
si_3e_2_TL |
| Sample type |
SRA |
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| Source name |
MCF10-A cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10-A
cell type: epithelial
treatment: eIF3e siRNA
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| Treatment protocol |
The cells were treated with 20nM si-control or si-eIF3e for 72h.
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| Growth protocol |
MCF-10a cells were cultured in DMEM/F12 medium with 0.5mg/mL hydrocortisone, 10µg/mL insulin, 20ng/mL EGF, 5% serum, 1X Pen/Strep, and incubate in 37℃ with 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The RNA were extracted with TRIzol reagent(thermo ambion, REF,15596018) and its standard protocol.
After clarification, 5 OD260 units of lysates were treated with 450 units of RNaseI (Ambion) for 45 min at RT. RPFs were isolated with Sephacryl S400 (GE, MicroSpin S-400 HR, 27514001). mRNAs and RPFs were isolated with TRIzol reagent(thermo ambion, REF,15596018). The rRNA removal for total mRNAs and RPFs was accomplished using Ribo-zero Gold (rRNA removal reagent(Human/Mouse/Rat)(illumina, REF, RZG1224) & Core kit(illumina, REF, MRZ11124C). For RPF, the extracted RNA was size-selected from 15% denaturing PAGE gels, cutting between 26-34 nt. An oligonucleotide adaptor was ligated to the 3’ end of footprints. Followed by reverse transcription, circularization and PCR amplification.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
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| Description |
total RNA
mRNA_counts.xlsx
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| Data processing |
Adapter sequences were trimmed from raw data using cutadapt 1.18 with parameters (-m 25 -M 35 --match-read-wildcards). Reads without adapter sequence were discarded.
Low-quality reads with Phred quality scores lower than 25 (>75% of bases) were removed using the fastx_quality_filter (http://hannonlab.cshl.edu/fastx_toolkit/).
Then sequence reads originating from rRNAs were identified and discarded by aligning the reads to human rRNA sequences downloaded from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) using Bowtie 1.1.2 with no mismatch allowed.
The remaining reads were mapped to the genome hg38 using STAR 2.5 with the following parameters: --runThreadN 8 --outFilterType Normal --outWigType wiggle --outWigStrand Stranded --outWigNorm RPM --alignEndsType EndToEnd --outFilterMismatchNmax 1 --outFilterMultimapNmax 1 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM GeneCounts --outSAMattributes.
Genome_build: hg38 (GRCh38)
Supplementary_files_format_and_content: The counts files were generated from alignment files by HTSeq-count in intersection-strict mode.
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| Submission date |
May 13, 2019 |
| Last update date |
Jun 01, 2020 |
| Contact name |
Xuerui Yang |
| E-mail(s) |
[email protected]
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| Organization name |
Tsinghua University
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| Department |
School of Life Sciences
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| Street address |
Haidian District
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| City |
Beijing |
| ZIP/Postal code |
100084 |
| Country |
China |
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| Platform ID |
GPL20795 |
| Series (1) |
| GSE131074 |
Ribosome profiling of eIF3e-KD MCF-10a cells |
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| Relations |
| BioSample |
SAMN11634336 |
| SRA |
SRX5823720 |
| 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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