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| Status |
Public on Aug 22, 2024 |
| Title |
Keratinocyte MPRA rep 2 [kc_2] |
| Sample type |
SRA |
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| Source name |
Keratinocyte MPRA
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Neonatal foreskin derived keratinocytes
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| Growth protocol |
Cells were grown in Keratinocyte-SFM and Medium 154 with pen/strep and antimycotic. Cells were infected with 16ul 50x virus per 200k cells with 8ug/ml polybrene. At least 12M cells were reverse infected, plated onto 15cm plates and selected with 1.5ug/ml puromycin. Cells were harvested in Qiagen Buffer RLT Plus.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA and DNA representing roughly 12 million cells were isolated using Qiagen’s AllPrep DNA/RNA Mini Kit. mRNA was then isolated from the total RNA using Dynabeads mRNA DIRECT purification kit (Thermo Fisher)
Reactions containing 500ng mRNA and 100nM of a gene-specific primer containing a sample index and UMI (GGW610-501 – GGW610-508, Table S5) were reverse transcribed using Superscript IV (Thermo Fisher), along with a no reverse transcriptase control. Reactions were incubated with 1ul thermolabile exonuclease I (NEB) for 10 min at 37C, followed by heat inactivation for 5min at 85C. The cDNA was cleaned with Ampure XP beads (Beckman Coulter) at a ratio of 1:1.1 sample to beads and eluted in the initial volume. The cDNA was amplified in 50ul reactions containing 5ul cDNA, LK612 at 80nM, one of LK134-701-LK134-712 at 80nM (Table S5), SYBR green (Thermo Fisher), and PrimeStar Max DNA Polymerase (Takara Bio). The reactions were amplified until they were just beginning to rise, then they were pooled and concentrated. The libraries were then gel purified and quantified using the KAPA library quantification kit (Roche). The 42 samples were put into 3 pools which were sequenced using an Illumina NextSeq Sequencer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
kc_RNA_2_S10
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| Data processing |
library strategy: Massively parallel reporter assay (MPRA)
Read 1 was unzipped and trimmed to the first 20bp containing the barcode using fastx_trimmer (fastx_trimmer -f 1 -l 20 -Q33)
Reverse complement of barcode was taken (fastx_reverse_complement -Q33)
UMI was extracted from index 2 (umi_tools extract --bc-pattern=NNNNNNNNNNNN)
Reads were aligned to the barcode library using bowtie2 (bowtie2 -p 8 -k 1 --norc -x)
Sam file was converted to a bam file (samtools view -S -b)
Bam file was sorted and indexed (samtools sort, samtools index)
Reads were deduplicated (umi_tools dedup -I --out-sam --no-sort-output)
File was converted to a tsv (awk '$1 !~ /^@/ {print $1"\t"$3}')
Counts per barcode were tabulated (umi_tools count_tab)
Genome_build: NA
Supplementary_files_format_and_content: Tab-delimited files with barcode counts for each oligo in each condition
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| Submission date |
Sep 02, 2021 |
| Last update date |
Aug 22, 2024 |
| Contact name |
Paul Khavari |
| Organization name |
Stanford University
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| Department |
Dermatology
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| Lab |
Khavari Lab
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| Street address |
269 Campus Drive, CCSR, Room 2150
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| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE183301 |
Inherited functional regulatory risk variants for common human cancers [MPRA] |
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| Relations |
| BioSample |
SAMN21208699 |
| SRA |
SRX11998840 |
| 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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