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| Status |
Public on Feb 04, 2022 |
| Title |
HEK293T rna-seq from C_2A15_2 |
| Sample type |
SRA |
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| Source name |
HEK293 Flp-In T-REx embryonic kidney cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
genotype/variation: HDLBP knockout guide 1
cellular compartment: Cytosol
replicate: replicate 2
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| Growth protocol |
HEK293T cells were grown in DMEM + 10%FCS
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cell fractionation by sequential detergent extraction was performed as previously described (Jagannathan et al., 2011, doi: 10.1007/978-1-61779-005-8_19 ) with minor modifications. HEK293 and HEK293 HDLBP knockout cell lines (1 15 cm dish per replicate) were grown to ~90 % confluency. Cells were washed with PBS. All further steps were carried out on ice using ice cold reagents and cells were always pelleted at 3000 g for 5 min at 4°C. First, PBS containing 50 µg/ml cycloheximide was added for 10 min. In the meantime cells were scraped using a rubber policeman. After pelleting, the cells were resuspended with 500 µl permeabilization buffer (110 mM KOAc, 25 mM K-HEPES [pH 7. 2], 2.5 mM Mg(OAc)2, 1 mM EGTA, 0.015 % digitonin, 1 mM DTT, 50 µg/ml cycloheximide, complete EDTA-free protease inhibitor cocktail [Roche], 40 U/mL SUPERaseIn [Thermo Fisher Scientifc]) per sample and incubated for 15 min at 4°C on a rotating wheel. After centrifugation the supernatant was collected as the cytosolic fraction. To wash the pellet, 5 ml of washing buffer (110 mM KOAc, 25 mM K-HEPES [pH 7.2], 2.5 mM Mg(OAc)2, 1mM EGTA, 0.004% digitonin, 1 mM DTT, 50 μg/ml cycloheximide) was used. After pelleting, 500 µl lysis buffer (400 mM KOAc, 25 mM K-HEPES [pH 7.2], 15 mM Mg(OAc)2, 0.5 % IGEPAL CA-630, 1 mM DTT, 50 μg/ml cycloheximide, complete EDTA-free protease inhibitor cocktail, 40 U/mL SUPERase•In) per sample was added for 5 min. After centrifugation, the supernatant was collected as the membrane fraction. The remaining pellet was resuspended with 500 µl lysis buffer, 200 µl 10 % sucrose in lysis buffer was added and the sample were centrifuged at 14000 rpm for 5 min at 4°C. The supernatant was removed, 200 µl wash buffer was added. After centrifucation the supernantant was remvoed and the pellet was collected as the nucleus fraction. Cytosol and membrane fractions were clarified by centrifugation at 7500 g for 10 min at 4°C. 250 µl of each sample was collected and RNA was isolated using Trizol LS (Thermo Fisher Scientific) in combination with RNA Clean & Concentrator-25 kit (Zymo Research) for RNA sequencing.
Illumina TruSeq Stranded mRNA Kit
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
RNA-Seq paired end
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| Data processing |
Basecalls were converted to fastq files using Bcl2Fastq (2.16.0.10). Reads were demultiplexed and 3’adapter sequences removed by Flexbar (v2.5). Read counts per gene and TPM values were obtained by RSEM (v.1.2.20) (Li and Dewey, 2011) using default parameters and Bowtie (v.1.1.2) (Langmead et al., 2009) as transcriptome alignment program.
Raw read counts were normalized using DESeq2 (Love et al., 2014) and pairwise comparisons between and within fractions performed using standard parameters.
Genome_build: hg19
Supplementary_files_format_and_content: processed_data_rnaseq_fractionation.txt: tab delimited matrix of read counts per gene for all samples, log2-transformed fold changes and p-adjusted values from DESeq2 output from pairwise comparisons, as well as normalized read counts per gene (norm. prefix).
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| Submission date |
Apr 07, 2020 |
| Last update date |
Feb 04, 2022 |
| Contact name |
Markus Landthaler |
| E-mail(s) |
markus.landthaler@mdc-berlin.de
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| Organization name |
Max Delbrueck Center
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| Department |
Berlin Institute for Medical Systems Biology
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| Street address |
Hannoversche Str. 28
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| City |
Berlin |
| ZIP/Postal code |
10115 |
| Country |
Germany |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE148260 |
Vigilin/HDLBP promotes translation of endoplasmic reticulum-targeted mRNAs (Cell fractionation RNA-seq) |
| GSE148262 |
HDLBP binds ER-targeted mRNAs by multivalent interactions to promote protein synthesis of transmembrane and secreted proteins |
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| Relations |
| BioSample |
SAMN14554478 |
| SRA |
SRX8077295 |
| 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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