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| Status |
Public on Dec 27, 2024 |
| Title |
RNA, nohs, siSrp72, rep2 |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Mus musculus |
| Characteristics |
tissue: cell line
cell line: NIH3T3
cell type: fibroblast
genotype: wt
treatment: no hs, siSrp72
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| Treatment protocol |
Briefly, 800 x 103 NIH3T3 cells were plated per well in a 6 well plate and transfected either with siScr or siSrp72. 24h later medium was replaced, and transfection was repeated. 24h later medium was removed, and cells were exposed to heat shock (45 ̊C, 10min) or not (37 ̊C, 10min) in 1ml medium pre-warmed to 37 ̊C or 45 ̊C.
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| Growth protocol |
NIH3T3 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum, 1% Penicilin- Streptomycin-Neomycin (PSN) antibiotic mixture and 1% non-essential amino acids from GibcoTM.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
After the treatment, plates were placed on ice, medium was removed, and the cells were washed in 2ml per well in cold 1X PBS without cycloheximide (CHX). PBS was removed and cells were lysed in 450μl of lysis buffer. (*Polysome buffer: 20mM Tris pH 7.5, 150mM NaCl, 100μg/ml CHX, 5mM MgCl2, 1mM DTT. *Lysis buffer: Polysome buffer + 25U/ml TURBOTM DNase + 1% Triton X-100. Cells were spun for 10min at 20000g and supernatant was collected.
mRNA was isolated from 100-200 ng of total RNA with NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490S). cDNA libraries were prepared according to the protocol for NEBNext® UltraTM II Directional RNA Library Prep Kit for Illumina (NEB, E7760S). Libraries were amplified 12 cycles and sent for Illumina sequencing. Experiment was performed using 2 biological replicates.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
BB3-6
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| Data processing |
Preprocessing for the raw fastq files including adaptor sequence removal was performed by Trim Galore v0.4.1 software
Preprocessed fastq files were aligned to mm10 genome by TopHat v2.0.10 with the following parameters: --read-edit-dist 3 --read-mismatches 3 --read-gap-length 3 --b2-very-sensitive --mate-inner-dist 100 --mate-std-dev 100
DEseq2 (Love et al., 2014), featureCounts (Liao et al., 2014), and enhancedVolcano were used for analyzing the differentially expressed genes.
Assembly: mm10
Supplementary files format and content: Bigwig coverage files were generated by using bamCoverage software with the parameter --normalizeUsingRPKM
Supplementary files format and content: read count table for all the RNA-seq samples
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| Submission date |
Sep 14, 2023 |
| Last update date |
Dec 27, 2024 |
| Contact name |
Hun-Goo Lee |
| Organization name |
Mass General Hospital
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| Department |
Molecular Biology
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| Lab |
Jeannie T Lee Lab
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| Street address |
185 Cambridge St
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE243284 |
7SL RNA and SRP orchestrate a global cellular response to acute thermal stress [RNA-seq] |
| GSE243286 |
7SL RNA and SRP orchestrate a global cellular response to acute thermal stress |
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| Relations |
| BioSample |
SAMN37398966 |
| SRA |
SRX21781380 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7782796_BB3-RNA6_nohs-siSrp72-2.mm10.b1.RPKM.bw |
52.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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