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Status |
Public on Jun 14, 2024 |
Title |
SDS22 +Dox/IAA biol rep 3 |
Sample type |
SRA |
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Source name |
HCT116
|
Organism |
Homo sapiens |
Characteristics |
cell line: HCT116 genotype: HCT116 Tet-OsTIR1 SDS22 WT (SDS22) treatment: Dox/IAA
|
Treatment protocol |
Cells were treated for 48h with 2 µg/ml doxycycline and 500 µM IAA
|
Growth protocol |
Cells were grown in low glucose-DMEM medium supplemented with 10% FBS and penicilline/streptomycin
|
Extracted molecule |
total RNA |
Extraction protocol |
RNA was extracted with the Mammalian Total RNA Miniprep kit (GenElute from SIGMA) RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent). Per sample, an amount of 1 microgram of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: Part # 15031047 Rev. E - October 2013) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single ‘A’ base to the 3’ ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence-libraries of each sample were equimolarly pooled and sequenced on an Illumina NextSeq 500 instrument (High Output, 75 bp, Single Reads, v2) at the VIB Nucleomics core (www.nucleomics.be).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
|
|
Description |
condition 4 - SDS22 degron treated SDS22@Dox@IAA@3@S23
|
Data processing |
Sequencing: Sequence-libraries of each sample were equimolarly pooled and sequenced on Illumina NextSeq 500 (76 bp, Single Reads, v1.5) at the VIB Nucleomics Core (www.nucleomics.be). Preprocessing: Low-quality ends and adapter sequences were trimmed off from the Illumina reads with FastX 0.0.14 and Cutadapt 1.15. Subsequently, small reads (length < 35 bp), polyA-reads (more than 90 % of the bases equal A), ambiguous reads (containing N), low-quality reads (more than 50 % of the bases < Q25) and artifact reads (all but three bases in the read equal one base type) were filtered using FastX 0.0.14 and ShortRead 1.36.1. With Bowtie2 2.3.3.1 we identified and removed reads that align to phix_illumina. Mapping: The preprocessed reads were aligned with STAR aligner v2.5.2b to the reference genome of Homo sapiens (GRCh38). Default STAR aligner parameter settings were used, except for ‘--outSAMprimaryFlag OneBestScore --twopassMode Basic --alignIntronMin 50 --alignIntronMax 500000 --outSAMtype BAM SortedByCoordinate’. Using Samtools 1.5, reads with a mapping quality smaller than 20 were removed from the alignments. Counting: The number of reads in the alignments that overlap with gene features was counted with featureCounts 1.5.3. Following parameters were chosen: -Q 0 -s 2 -t exon -g gene_id. Assembly: Homo sapiens Ensembl GRCh38.88 Supplementary files format and content: Matrix table with raw gene counts for every gene and every sample
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|
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Submission date |
Dec 06, 2023 |
Last update date |
Jun 14, 2024 |
Contact name |
Rekin's Janky |
E-mail(s) |
Nucleomics.Bioinformatics@vib.be
|
Organization name |
VIB
|
Department |
Nucleomics Core
|
Street address |
Herestraat 49 Box 816
|
City |
Leuven |
ZIP/Postal code |
B-3000 |
Country |
Belgium |
|
|
Platform ID |
GPL18573 |
Series (1) |
GSE249519 |
SDS22 coordinates the assembly of holoenzymes from nascent protein phosphatase-1 |
|
Relations |
BioSample |
SAMN38698368 |
SRA |
SRX22805404 |