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| Status |
Public on Apr 25, 2024 |
| Title |
Sample 5_HeLa cells, 80S footprints, eIF3d KD, rep2 |
| Sample type |
SRA |
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| Source name |
HeLa
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
cell type: human epithelial cell
genotype: EIF3D knock down
treatment: eIF3d siRNA, 72 hours
molecule subtype: 80S RNA footprints
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| Treatment protocol |
Sample1 -Sample12:
Twenty-four hours after seeding, cells were transfected with the ON-TARGETplus siRNA cocktail system from Dharmacon at a final concentration of 5 nM. INTERFERin (Polyplus) was used as a transfection reagent, 100 ul were used per dish. Cells were harvested 72 hours after transfection at approximately 80% confluency.
Sample13-Sample28:
Twenty-four hours after seeding, cells were transfected with the ON-TARGETplus siRNA cocktail system from Dharmacon at a final concentration of 5 nM. INTERFERin (Polyplus) was used as a transfection reagent, 100 ul were used per dish. Cells were harvested 72 hours after transfection at approximately 80% confluency.
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| Growth protocol |
Sample1 -Sample12:
HeLa cells were grown at 37 °C and 5% CO2 in Ø 15 cm dishes in DMEM supplemented with 10% FBS.
Sample13-Sample28:
HeLa cells were grown at 37 °C and 5% CO2 in Ø 15 cm dishes in DMEM supplemented with 10% FBS.
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| Extracted molecule |
other |
| Extraction protocol |
Sample1 -Sample12:
For footprint libraries cycloheximide was added to a final concentration of 100 µg/ml 1 min prior to harvesting. Cells were washed with ice cold 1x PBS and lysed in buffer A (10 mM HEPES [pH 7.5], 62.5 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 1mM PMSF, 1 µg/ml Aprotinin, 1 µg/ml Leupeptin, 1 µg/ml Pepstatin, Complete Mini EDTA-free [Roche] – 1 tablet/5 ml, 1% Triton X-100, 100 µg/ml Cycloheximide) on the dish. The resulting lysate was collected and cleared by centrifugation. Aliquots of twelve absorbance units (AU) OD260 of WCE were flash frozen in liquid nitrogen and used for footprint isolation. One aliquot of 12 AU OD260 was thawed on ice, digested with 5 µl RNAseI (Ambion) for 30 min at 24 °C with mild shaking (300 rpm) and inactivated with SuperaseIN (Ambion). 80S ribosomes were separated by high-velocity sedimentation through a 5% to 45% sucrose gradient at 39,000 rpm for 2.5 h using the SW41Ti rotor. The gradients were scanned at 254 nm to visualize the ribosomal species and fractions containing 80S ribosomes were collected and mixed with RNA Blue (TopBio). RNA was isolated according to vendor’s instructions, FP RNA was size selected on 15% TBE-Urea PAGE (Biorad) in range 20-40 nt.
Sample13-Sample28:
For mRNA libraries the growth media was aspirated and cells were harvested into 5 ml of RNA Blue per Ø 15 cm dish. RNA was isolated according to vendor’s instructions. mRNA was isolated from the total RNA using Poly(A)purist MAG kit (Ambion), fragmented using RNA Fragmentation Reagents (Ambion) and size selected on 15% TBE-Urea PAGE in range 50-100 nt.
Sample1 -Sample12:
According to Ingolia et al., 2010, Methods Enzymology
Sample13-Sample28:
library was prepared using SMARTer smRNA-Seq Kit (TAKARA) according to vendor’s instructions
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
library strategy: Ribo-seq
Sample1 -Sample12:
Adapter sequence “CTGTAGGCACCATCAAT” and all sequences 3’ of the adapter were trimmed from the 3’ end of each read; flanking Ns were trimmed from both 5' and 3' ends of each read; and reads without adapter sequence and reads shorter than 15 nt after trimming were discarded by Cutadapt v. 3.5
rRNA and tRNA reads were discarded after alignment with Bowtie2 v. 2.4.4. Used sequences rRNA: RNA5S1, RNA5-8SN5, RNA18SN5 and RNA28SN5 from NCBI, and ENSG00000211459 and ENSG00000210082 from Ensembl. Used sequences tRNA: human tRNA sequences from tRNAdb and all high confidence hg38 tRNA sequences from GtRNAdb.
Reads were aligned to genome and transcriptome from Ensembl (GRCh38.p13, annotation release 104) by the STAR aligner v. 2.7.9a_2021-06-25 using local alignment with 10% mismatches, and with indels and soft clipping allowed for transcriptomic alignment
Alignments were indexed using Samtools index v. 1.13
Read counts within CDS region were counted with Htseq-count v. 0.13.5
Sample13-Sample28:
First 3 nucleotides on the 5’ end were removed, and poly(A) sequence (10xA) was trimmed from the 3’end; flanking Ns were trimmed from both 5' and 3' ends of each read; and reads without adapter sequence and reads shorter than 15 nt after trimming were discarded by Cutadapt v. 3.5
rRNA and tRNA reads were discarded after alignment with Bowtie2 v. 2.4.4. Used sequences rRNA: RNA5S1, RNA5-8SN5, RNA18SN5 and RNA28SN5 from NCBI, and ENSG00000211459 and ENSG00000210082 from Ensembl. Used sequences tRNA: human tRNA sequences from tRNAdb and all high confidence hg38 tRNA sequences from GtRNAdb.
Reads were aligned to genome and transcriptome from Ensembl (GRCh38.p13, annotation release 104) by the STAR aligner v. 2.7.9a_2021-06-25 using local alignment with 10% mismatches, and with indels and soft clipping allowed for transcriptomic alignment
Alignments were indexed using Samtools index v. 1.13
Read counts within CDS region were counted with Htseq-count v. 0.13.5
Assembly: GRCh38.p13, annotation release 104
Supplementary files format and content: Ensembl gene identifiers and gene counts in tab-separated values file format
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| Submission date |
Nov 01, 2022 |
| Last update date |
Apr 25, 2024 |
| Contact name |
Anna Herrmannová |
| Organization name |
Institute of Microbiology of the Czech Academy of Sciences
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| Lab |
Laboratory of Regulation of Gene Expression
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| Street address |
Vídeňská 1083
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| City |
Prague |
| ZIP/Postal code |
142 20 |
| Country |
Czech Republic |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE216967 |
Differential expression analysis connects knock-downs of eIF3e and eIF3d with the MAP kinase signaling pathway |
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| Relations |
| BioSample |
SAMN31544954 |
| SRA |
SRX18102690 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6701454_192111754_d_FP_II_R1-a17m15t-rRNA-tRNA-r01M1s_Aligned.sortedByCoord.out-Ensembl.tbl.txt.gz |
80.2 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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