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| Status |
Public on Feb 21, 2020 |
| Title |
Plasmid-Baseline |
| Sample type |
SRA |
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| Source name |
MIN6 mouse beta cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: MIN6
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| Treatment protocol |
10 million MIN6 cells were seeded in each of seven 15 cm2 dishes. The cells were 60-70% confluent the next day. Each 15 cm2 dish was replaced with 20 ml of fresh media, and transfected with 7ug of the MPRA plasmid library using 55ul Lipofectamine 2000 (38% transfection efficiency). Six hours after transfection, media was either i) not changed (MPRA under standard culture conditions), ii) replaced with media containing 250 nM Thapsigargin dissolved in 0.025% DMSO, or iii) replaced with media containing 0.025% DMSO. Thirty hours after transfection, cells were trypsinized and collected by centrifugation. Cell pellets were frozen at -80°C. For each condition (standard culture / DMSO / 250 nM TG), MIN6 cells were transfected on five separate days to generate biological replicates.
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| Growth protocol |
MIN6 were grown in DMEM (4.5 g/L glucose; Life Technologies) supplemented with 1 mM sodium pyruvate (Life Technologies), 100 μM 2-mercaptoethanol (Sigma), and 10% fetal bovine serum (Seradigm).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was extracted from frozen cell pellets using the Qiagen RNeasy Midi kit. Following DNase treatment, a mixture of 3 GFP-specific biotinylated primers (Supplementary Table 1; #120, #123 and #126) were used to immunoprecipitated GFP transcripts using Streptavidin C1 Dynabeads (Life Technologies). Following another round of DNase treatment, cDNA was synthesized from GFP mRNA using SuperScript IV and purified with AMPure XP beads. Quantitative PCR using primers specific for GFP (Supplementary Table 1; #34 and #52) was used to determine the cycle at which linear amplification begins for each replicate. Replicates were diluted to approximately the same concentration based on the qPCR results, and PCR with primers #34 and #52 was used to amplify barcodes associated with the ~13.5k sequences included in the MRPA library for each replicate (9 cycles for standard culture, and 13 cycles for DMSO / 250 nM TG). A second round of PCR (6 cycles) was used to add Illumina sequencing adaptors to the DNA/RNA replicates. The resulting MPRA barcode libraries were spiked with 5% PhiX and sequenced using Illumina single-end 31 bp chemistry (with 8 bp index read), clustered at 80-90% maximum density.
Oligos were synthesized (Agilent Technologies) as 230 bp sequences containing 200 bp of genomic sequences and 15 bp of adaptor sequence on either end. Unique 20 bp barcodes were added by PCR along with additional constant sequence for subsequent incorporation into a backbone vector by Gibson assembly. The oligo library was expanded by electroporation into E. coli, and the resulting plasmid library was sequenced by Illumina 2 X 150 bp chemistry to acquire oligo-barcode pairings. The library underwent restriction digestion, and GFP with a minimal TATA promoter was inserted by Gibson assembly resulting in the 200 bp oligo sequence positioned directly upstream of the promoter and the 20 bp barcode falling in the 3’ UTR of GFP. After expansion within E. coli the final MPRA plasmid library was sequenced by Illumina 1 X 31 bp chemistry to acquire a baseline representation of each oligo-barcode pair within the library. Note: 2 separate batches of the MPRA library were prepared. The first batch was used to perform MPRA under standard culture conditions. This MPRA library was then electroporated into E. coli to obtain a second batch of the MRPA library, which was used for the DMSO-TG experiments.
Massively parallel reporter assay (MPRA)
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
MIN6_MPRA_Baseline_FinalMatrix.out
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| Data processing |
The sum of the barcode counts for each oligo within replicates was median normalized, and oligos showing differential expression relative to the plasmid input were identified by modeling a negative binomial distribution with DESeq2 and applying a false discovery rate (FDR) threshold of 1%.
For sequences that displayed significant MPRA activity, a paired t-test was applied on the log-transformed RNA/plasmid ratios for each experimental replicate to test whether the reference and alternate allele had similar activity. An FDR threshold of 10% was used to identify SNPs with a significant skew in MPRA activity between alleles (allelic skew). Because the MPRA testing standard culture conditions was performed with a separate MRPA library preparation. Therefore, the DMSO-TG MPRA results were not directly compared to MPRA performed under standard culture conditions.
Genome_build: Custom; T2D_khetan_All_20180111.asi.probes.fa
Supplementary_files_format_and_content: txt
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| Submission date |
Feb 20, 2020 |
| Last update date |
Sep 14, 2020 |
| Contact name |
Shubham Khetan |
| E-mail(s) |
skhetan@bwh.harvard.edu
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| Phone |
8607943361
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| Organization name |
Brigham and Women's Hospital
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE145643 |
Functional characterization of thousands of type 2 diabetes-associated and chromatin-modulating variants under steady state and endoplasmic reticulum stress |
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| Relations |
| BioSample |
SAMN14146629 |
| SRA |
SRX7760653 |
| 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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