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| Status |
Public on May 03, 2018 |
| Title |
mcherry_unmod_1 |
| Sample type |
SRA |
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| Source name |
mCherry 200,000 member 5' UTR library
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HEK293T
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| Treatment protocol |
HEK293T cells were plated on 10cm cell culture dishes 24 hours before transfection (1 - 2 million per plate). At 60% to 80% confluency, cells were transfected with 14.5 µg of library mRNA using Lipofectamine MessengerMAX (Thermo Fisher Scientific) following the manufacturer’s protocol. Washed plates with 10 ml 1x DBPS and 10 ml media (DMEM with 10% FBS and 1% Penicillin-Streptomycin) after one hour of incubation. Cells were lysed 12 hours after transfection.
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| Growth protocol |
HEK293T cells were cultured on ECM-coated plates in Dubelco's DMEM media supplemented with FBS (5%), penicillin (.05%), and streptomycin (.05%)
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| Extracted molecule |
total RNA |
| Extraction protocol |
Salt solution (10x): 100 mM NaCl, 100 mM MgCl2,100 mM Tris-HCl pH 7.5, and RNase-free water (Zuccotti P, Modelska A 2016). Wash buffer: 100 µg/ml cycloheximide in RNase-free DPBS (10 ml per plate). Lysis buffer: 1x salt solution, 1% of 20% Triton X-100, 1 mM DTT, 0.2 U/µl SUPERase-In (Thermo Fisher Scientific), 100 µg/ml cycloheximide. Wash buffer and lysis buffer were chilled throughout the protocol. After 12 hours of growth at 37 °C, cells were placed on ice and media was aspirated. Translating ribosomes were halted by adding 5 ml of wash buffer and were then placed at 37 °C for 5 minutes followed by aspiration on ice. Washed by adding 5 ml wash buffer and aspirating thoroughly. Added 300 µl of ice-cold lysis buffer, scraped cells, broke up cell clumps by pipetting ~5 times, and then placed suspended cells into a pre-chilled microcentrifuge tube. Let sit for 10 minutes on ice and then triturated by passing through a 25-G needle 10 times [1]. Spun at 16,000 x g for 5 minutes to pellet cell debris and nuclei. Saved supernatant and added 1.5 µl of 1 U/µl DNase I (final concentration of 0.005 U/µl) and placed on ice for 30 minutes. Either stored at -80 °C or proceeded directly to polysome profiling.
For both the eGFP and mCherry library, randomized 50-mer oligos were Gibson assembled to serve as 5' UTRs for each CDS. To create the designed library, oligos were made via oligo synthesis array (CustomArray Inc.) and cloned into the eGFP vector.For all libraries, 25 nucleotides of defined sequence preceed the inserted 5' UTRs.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
After sequencing, RNA from each polysome fraction was identified by fraction-specific barcodes
cutadapt --length 59 --minimum-length 59 -o r1.fastq -p r2.fastq
Combine r1.fastq sequences (UTRs) and r2.fastq (UMIs) into a single CSV for Bartender
bartender_single_com -f input_csv -o output_file.csv -c 2 -d 2 -z -1
Calculate relative reads within fractions and then, using these values, calculate relative reads per fraction for a given UTR. Calculate mean ribosome load (MRL) by multiplying each fraction’s relative reads by the number of ribosomes associated with each fraction and then sum the values.
Supplementary_files_format_and_content: CSV files contain 5' UTR sequences, read counts per fraction, relative counts, and MRL ('rl').
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| Submission date |
May 03, 2018 |
| Last update date |
May 04, 2018 |
| Contact name |
Paul J Sample |
| E-mail(s) |
pjsample@gmail.com
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| Organization name |
University of Washington
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| Department |
Electrical Engineering
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| Street address |
West Stevens Way NE
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| City |
Seattle |
| State/province |
Washington |
| ZIP/Postal code |
98195 |
| Country |
USA |
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| Platform ID |
GPL21697 |
| Series (1) |
| GSE114002 |
Human 5′ UTR design and variant effect prediction from a massively parallel translation assay |
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| Relations |
| BioSample |
SAMN09045422 |
| SRA |
SRX4035640 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3130441_mcherry_1.csv.gz |
23.5 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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