|
| Status |
Public on May 09, 2021 |
| Title |
HEK293T-RBFOX2- STAMP 72h high rep2 |
| Sample type |
SRA |
| |
|
| Source name |
HEK293T
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
|
| Treatment protocol |
For STAMP fusion protein expression STAMP stable HEK293T cells were induced with 50ng/ml (low) or 1ug/ml (high) doxycycline in DMEM for 24-72 hours, followed by Trizol extraction and Direct-zol miniprep (Zymo Research) column purification in accordance with manufacturer protocol. Uninduced cells of the same genetic background were used as negative controls. For mTOR perturbation experiments, cells were treated with 100nM Torin-1 or 100nM insulin alongside 1ug/ml doxycycline induction and harvested for RNA after 72 hours 37C incubation.
|
| Growth protocol |
All stable STAMP HEK293T cell lines were generated using human lenti-X HEK293T cells (HEK293XT, Takara Bio) which are derived from transformed female human embryonic kidney tissue. Cells were maintained in DMEM (4.5 g/L D-glucose) supplemented with 10% FBS (Gibco) at 37o C with 5% CO2. Cells were periodically passaged once at 70-90% confluency by dissociating with TrypLE Express Enzyme (Gibco) at a ratio of 1:10.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Trizol extraction (Fisher) and Direct-zol miniprep (Zymo Research) column purification in accordance with manufacturer protocol
Illumina TruSeq stranded mRNA or total RNA
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
Sample28_S21_merged
|
| Data processing |
Raw reads were trimmed using cutadapt (v1.14) using the following parameters -O 5 -f fastq --match-read-wildcards --times 2 -e 0.0 --quality-cutoff 6 -m 18 -o data.fastqTr.fq -b TCGTATGCCGTCTTCTGCTTG -b ATCTCGTATGCCGTCTTCTGCTTG -b CGACAGGTTCAGAGTTCTACAGTCCGACGATC -b GATCGGAAGAGCACACGTCTGAACTCCAGTCAC -b AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA -b TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT data.fastq.gz
Trimmed reads were mapped to and filtered of repeat elements (RepBase 18.05) with STAR (2.4.0i) using the following parameters: --alignEndsType EndToEnd --genomeDir repbase --genomeLoad NoSharedMemory --outBAMcompression 10 --outFileNamePrefix data --outFilterMultimapNmax 10 --outFilterMultimapScoreRange 1 --outFilterScoreMin 10 --outFilterType BySJout --outReadsUnmapped Fastx --outSAMattrRGline ID:foo --outSAMattributes All --outSAMmode Full --outSAMtype BAM Unsorted --outSAMunmapped Within --outStd Log --readFilesIn data.fastqTr.fq --runMode alignReads --runThreadN 8
Reads unmapped to repeat elements were mapped to the human genome with STAR using the same parameters as the previous step, using an hg19 index in place of the repeat element index
Subread featureCounts (-a gencode.v19.annotation.gtf -s 2 -p -o counts.txt data.bam) was used to count features using human annotations (Gencode v19)
Edits were called using SAILOR (http://github.com/yeolab/sailor) using default parameters and dbSNP (v147) to remove known SNPs.
bigwig files were generated from filtered BAM files (intermediates from SAILOR), using the following commands from BedTools (v2.27.1). : bedtools genomecov -split -strand - -g hg19.chrom.sizes -bg -ibam fwd.bam > fwd.bg bedtools sort -I fwd.bg > fwd.sorted.bg bedGraphToBigWig fwd.sorted.bg hg19.chrom.sizes fwd.sorted.bw bedtools genomecov -split -strand + -g hg19.chrom.sizes -bg -ibam rev.bam > rev.bg bedtools sort -I rev.bg > rev.sorted.bg bedGraphToBigWig rev.sorted.bg hg19.chrom.sizes rev.sorted.bw
Genome_build: hg19
|
| |
|
| Submission date |
Aug 04, 2020 |
| Last update date |
May 09, 2021 |
| Contact name |
Gene Yeo |
| E-mail(s) |
geneyeo@ucsd.edu
|
| Organization name |
UCSD
|
| Street address |
2880 Torrey Pines Scenic Dr. Room 3805/Yeo Lab
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE155649 |
Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes [RNA-seq] |
| GSE155729 |
Robust single-cell discovery of RNA targets of RNA binding proteins and ribosomes |
|
| Relations |
| BioSample |
SAMN15721340 |
| SRA |
SRX8880871 |
