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| Status |
Public on Oct 21, 2015 |
| Title |
A3SS_DNA |
| Sample type |
SRA |
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| Source name |
plasmid prep
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| Organism |
synthetic construct |
| Characteristics |
plasmid_library: A3SS_DNA (alternative 3' library)
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| Treatment protocol |
For transfection of a complex pool of plasmids, 1.2 million cells were seeded in a 10cm dish 24 hours before transfection. We mixed 10ug of the plasmid library in 1ml of Opti-MEM Reduced Serum Medium (Life Technologies) with 30ul of Lipofectamine LTX and 10ul of Plus Reagent (Life Technologies), before transfecting into the 10cm dish. The DMEM was was replaced 5 hours after transfection.
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| Growth protocol |
HEK293 cells were cultured in in DMEM (Cellgro) + 10% FBS and L-glutamine/penicillin/streptomycin on coated plates. Plates were coated for 24 hours with 8mL of 100x diluted extracellular matrix gel (Sigma-Aldrich) before HEK293 cells were added to the plates.
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| Extracted molecule |
other |
| Extraction protocol |
Total RNA was extracted using RNeasy (Qiagen) kits 24 hours after transfection. The optional on column DNaseI digest was performed with the RNase-Free DNase Set (Qiagen). Total RNA quality and purity was tested by measuring the A260/A280 ratio on a NanoDrop 1000 Spectrophotometer, and in some cases by measuring the ratio of the 18S and 28S rRNA bands on a native 1% agarose gel. mRNA was separated from 35-48ug total RNA using polyA Spin mRNA Isolation Kits (New England Biolabs). Isolated mRNA was again digested by DNaseI for 30 minutes using the Turbo DNA-free kit (Ambion). cDNA was then synthesized from 109-374ng mRNA using MultiScribe Reverse Transcriptase (Ambion) and Oligo d(T)16 (Ambion). cDNA synthesis was performed by holding reactions at 25°C for 10 min, 42°C for 110 min, and 85°C for 5 min. The quality of cDNA and presence of DNA contamination were checked through qPCR: Citrine, mCherry, and TBP were compared using cDNA, No Reverse Transcription Controls (NRTC) and a No Template Control (NTC). The results indicated that there was no plasmid or genomic DNA carryover into the cDNA reactions.
The resulting cDNA was then amplified by PCR to generate products compatible with the Illumina HiSeq2000 Flow Cell. PCR reactions were performed in 100uL with 2x Phusion HF Master Mix (New England Biolabs), 50pmol forward primer and 50pmol reverse primer with sample specific barcodes and 20% of each cDNA reaction. Cycling was done on a BioRad T100TM Thermal Cycler with the following protocol: 98°C for 5 min, then 7 cycles of 98°C for 10s, 67.5°C for 15s, 72°C for 30s, and a final extension step at 72°C for 5 min. The necessary number of cycles was determined for each sample by first running PCR reactions with EvaGreen in a Biorad CFX and determining when fluorescence began to plateau. Following PCR, 10% of the products were run on a 2% agarose gel to determine if the expected bands were present. The remainder of the PCR products was purified using the QIAquick PCR Purification kit (Qiagen) and eluted into 30uL of EB. Concentrations as well as A260/280 and A260/230 ratios were measured on a NanoDrop 1000 Spectrophotometer. Illumina compatible PCR products were also generated from the DNA plasmid library with the same protocol as above except the cDNA template was replaced with 10ng of plasmid library DNA and the PCR reaction was performed with 20 cycles.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
Plasmid DNA
synthetic sequences
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| Data processing |
The fastq files were processed using custom python scripts. All of the analysis is available at https://github.com/Alex-Rosenberg/cell-2015
Genome_build: NA
Supplementary_files_format_and_content: For each library we have a file with all the barcode – intronic sequence associations, as well as a matrix with the number of spliced reads at each position for each library member.
Supplementary_files_format_and_content: [A3SS_seq.txt] Sequences in alternative 3' library
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| Submission date |
Oct 19, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander B Rosenberg |
| E-mail(s) |
[email protected]
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| Organization name |
University of Washington
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| Department |
Electrical Engineering
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| Lab |
Seelig Lab
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| Street address |
4000 15th Ave NE
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| City |
Seattle, WA 98195 |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL17769 |
| Series (1) |
| GSE74070 |
Learning the Sequence Determinants of Alternative Splicing from Millions of Random Sequences |
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| Relations |
| BioSample |
SAMN04194111 |
| SRA |
SRX1353954 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1911083_A3SS_seq.txt.gz |
51.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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