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| Status |
Public on Feb 15, 2019 |
| Title |
F9-RNA-3: F9 transfection RNA replicate 3 |
| Sample type |
SRA |
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| Source name |
cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
cell type: Human epithelial liver cancer cell line
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| Treatment protocol |
For each experiment, about 5 million cells were plated in 15-cm plates and incubated for 24 hours before transfection. Each of three independent cultures (replicates) were transfected with 15 μg of the constructed MPRA libraries using X-tremeGENE HP (Roche 06366236001) for 24 hours (except for PKLR additionally transfected for 48 hours and MYC rs6983267 transfected for 32 hours, with 20nM LiCl being added after 24 hours). The ZRS library was co-transfected with HOXD13 as well as HOXD13 and HAND2 in two separate experiments.
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| Growth protocol |
HepG2, HEK293T, HeLa, HaCaT, Min6, Neuro-2a, LNCap, SK-MEL-28 were obtained from American Type Culture Collection, and primary gliobastoma (GBM) cell line SF7996 was obtained from UCSF (Dr. Costello’s lab). 2.0x10^5 cells were cultured in 96-well plates overnight using standard protocols. LNCaP was grown in media with 100nM DHT.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
After cells were harvested, genomic DNA and total RNA were extracted using AllPrep DNA/RNA mini kit (Qiagen 80204). Total RNA was subjected to mRNA selection (Oligotex, Qiagen 72022) and treated with Turbo DNase (Thermo Fisher Scientific AM2238) to remove contaminating DNA.
For each replicate, RNA was reverse transcribed with Superscript II (Invitrogen 18064-014) using a primer downstream of the tag, which contained a sample index and a P7 Illumina adaptor sequence. The resulting cDNA was first pooled and then split into multiple reactions to reduce PCR jackpotting effects. Amplification was performed with Kapa Robust polymerase (Roche KK5024) for three cycles, incorporating unique molecular identifiers (UMIs) 10 bp in length. PCR products were cleaned up with AMPure XP beads (Beckman Coulter A63880) to remove the primers and concentrate the products. These products underwent a second round of amplification in eight reactions per replicate for 15 cycles, with a reverse primer containing only P7. All reactions were pooled, run on an agarose gel for size-selection, and then sequenced.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Targeted barcode amplification incl. UMIs
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| Data processing |
Libraries were sequenced on MiSeq or NextSeq 500 instruments (Illumina) and base called using the instruments' Real Time Analysis software. Multiplexed paired end (PE) Illumina sequencing data was split based on sample barcodes allowing up to one substitution in the barcode sequence.
Paired-end reads shorter or equal to read length were consensus called and adapter trimmed.
Barcodes (consensus called paired-end reads) and UMI sequences containing unresolved bases (N) or not matching the designed length of 15bp were excluded. Each barcode x UMI pair was counted only once and only barcodes matching perfectly to those identified in the library assigment were considered.
Only barcodes observed in RNA and DNA of the same sample were considered.
Genome_build: GRCh37, GRCh38
Supplementary_files_format_and_content: Non-FASTA data files are in gzip-compressed text file formats. Files with the sufix variants.txt.gz contain an assignment of barcodes to sequence variants. Variant locations are described in a coordinate system that starts 20 bases up- und and downstream of the targeted region. FASTA files contain the wild-type sequence. DNA.tsv.gz and RNA.tsv.gz files contain assigned RNA and DNA barcode counts in tab-separated text format.
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| Submission date |
Feb 14, 2019 |
| Last update date |
Mar 01, 2019 |
| Contact name |
Jay Shendure |
| Organization name |
University of Washington
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| Department |
Genome Sciences
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| Lab |
Shendure
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| Street address |
3720 15th Ave NE
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195-5065 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE126550 |
Saturation mutagenesis of disease-associated regulatory elements |
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| Relations |
| BioSample |
SAMN10929106 |
| SRA |
SRX5444819 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3604169_F9-RNA-3.tsv.gz |
22.4 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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