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| Status |
Public on Feb 11, 2020 |
| Title |
transfection_GM12878_1_unstim |
| Sample type |
SRA |
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| Source name |
EBV transformed B cell line
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| Organism |
Homo sapiens |
| Characteristics |
stim condition: none
cell line: GM12878
vector delivery: transfection
replicate: 1
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| Biomaterial provider |
Coriell Cell Repositories https://www.coriell.org/0/Sections/Search/Sample_Detail.aspx?Ref=GM12878
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| Treatment protocol |
Jurkat T cells were stimulated with 2.5 ug/mL of anti-CD3 (Biolegend, 317304) and 10 ng/mL of PMA (Sigma, P1585-1MG) for 1 hour prior to harvesting. BJAB and GM12878 B cells were stimulated with 2.5 ug/ml of anti-IgM and 2 ug/ml anti-CD40 for 2 hours. U937 monocytes were stimulated with 100 ng/mL LPS for 2 hours.
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| Growth protocol |
BJAB, Jurkat, U937, and GM12878 cell lines were cultured using RPMI 1640 (ThermoFisher, 21870092) containing 10% fetal bovine serum (FBS, VWR, 97068-091; 20% for GM12878) with 1% Penn/strep (VWR, 45000-652), 1% L-Glutamine (ThermoFisher, 25030081), and 1% HEPES (Sigma, H0887-100ML). Cells were maintained at a culture density between 100K-1M cells/mL.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from cells using Qiagen Maxi RNeasy (Qiagen, 75162) following the manufacturer’s protocol including the on-column DNase digestion. A second DNase treatment was performed on the purified RNA using 5 μL of Turbo DNase (Life Technologies, AM2238) with buffer, in 750 μL of total volume for 1 hour at 37°C. The digestion was stopped with the addition of 7.5 μL 10% SDS and 75 μL of 0.5M EDTA followed by a 5 minute incubation at 70°C. The total reaction was then used for pulldown of GFP mRNA. Water was added to the DNase digested RNA to bring the total volume to 898 μL to which 900 μL of 20X SSC (Life Technologies, 15557-044), 1800 μL of Formamide (Life Technologies, AM9342) and 2 μL of 100 uM biotin-labeled GFP probe (GFP_BiotinCapture_1-3, IDT, Ext. Data Table 10) were added and incubated for 2.5 hours at 65°C. Biotin probes were captured using 400 μL of pre- washed Streptavidin beads (Life Technologies, 65001) eluted in 500 μL of 20X SSC. The hybridized RNA/probe bead mixture was agitated on a nutator at room temperature for 15 minutes. Beads were captured by magnet and washed once with 1x SSC and twice with 0.1x SSC. Elution of RNA was performed by the addition of 25 μL water and heating of the water/bead mixture for 2 minutes at 70°C followed by immediate collection of eluent on a magnet. A second elution was performed by incubating the beads with an additional 25 μL of water at 80°C. A final DNase treatment was performed in 50 μL total volume using 1 μL of Turbo DNase with addition of the buffer incubated for 60 minutes at 37°C followed by inactivation with 1 μL of 10% SDS and purification using RNA clean SPRI beads (Beckman Coulter, A63987).
First-strand cDNA was synthesized from half of the DNase-treated GFP mRNA with SuperScript III and a primer specific to the 3’ UTR (MPRA_v3_Amp2Sc_R) using the manufacturer’s recommended protocol, modifying the total reaction volume to 40 μL and performing the elongation step at 47°C for 80 minutes. Single-stranded cDNA was purified by SPRI and eluted in 30 μL EB. To minimize amplification bias during the creation of cDNA tag sequencing libraries, samples were amplified by qPCR to estimate relative concentrations of GFP cDNA using 1 μL of sample in a 10 μL PCR reaction containing 5 μL Q5 NEBNext master mix, 1.7 μL Sybr green I diluted 1:10,000 (Life Technologies, S-7567) and 0.5 uM of TruSeq_Universal_Adapter and MPRA_Illumina_GFP_F primers (Ext. Data Table 10). Samples were amplified with the following qPCR conditions: 95°C for 20 seconds, 40 cycles (95°C for 20 sec, 65°C for 20 sec, 72°C for 30 sec), 72°C for 2 min. The number of cycles for sample amplification was 1-n (the number of cycles it took for each sample to pass the threshold) from the qPCR. To add Illumina sequencing adapters, 10 uL of cDNA samples and mpra:pTNFAIP3:gfp plasmid control (diluted to the qPCR cycle range of the samples) were amplified using the reaction conditions from the qPCR scaled to 50 uL, excluding Sybrgreen I. Amplified cDNA was SPRI purified and eluted in 40 μL of EB. Individual sequencing barcodes were added to each sample by amplifying the entire 40 μL elution in a 100 μL Q5 NEBNext reaction with 0.5 uM of TruSeq_Universal_Adapter primer and a reverse primer containing a unique 8 bp index (Illumina_Multiplex, Ext. Data Table 10) for sample demultiplexing post-sequencing. Samples were amplified at 95°C for 20 seconds, 6 cycles (95°C for 20 sec, 64°C for 30 sec, 72°C for 30 sec), 72°C for 2 minutes. Indexed libraries were SPRI purified and pooled according to molar estimates from Agilent TapeStation quantifications. Samples were sequenced using 1x30 bp chemistry on an Illumina HiSeq 2500 or NextSeq. To determine oligo/barcode combinations within the MPRA pool, Illumina libraries were prepared from the mpra∆orf plasmid library by performing 4 separate amplifications with 200 ng of plasmid in a 100 μL Q5 NEBNext PCR reaction containing 0.5 uM of TruSeq_Universal_Adapter and MPRA_v3_TruSeq_Amp2Sa_F primers with the following conditions: 95°C for 20 sec, 6 cycles (95°C for 20 sec, 62°C for 15 sec, 72°C for 30 sec), 72°C for 2 minutes. Amplified material was SPRI purified using a 0.6x bead/sample ratio and eluted with 30 μL of EB. Sequencing indexes were then attached using 20 μL of the eluted product and the same reaction conditions as for the tag-seq protocol, except the number of enrichment cycles was lowered to 5. Samples were molar pooled and sequenced using 2x150 bp chemistry on Illumina HiSeq 2500 and NextSeq.
MPRA library preparation
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
20190823_MPRA_tag_count_matrix.txt.gz
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| Data processing |
A bowtie index of the ordered probe sequences and 16Ns for the barcode was created and the long reads for mapping barcodes to enhancers aligned to this index using Bowtie2. Associated barcodes were extracted and classified into those that map uniquely to a specific error-free probe, and those that do not (probe_barcode_association_3.4E6_2.map.gz).
MPRA RNA output and DNA input sequencing reads were mapped to the known tag sequences using a custom python script (quantifyRNATags.py), allowing for up to four mismatches within the constant region (the common sequence before the tag sequence) and no mismatches within the tag sequence.
The barcode counts were input, and tags having fewer than 30 reads in the input (DNA) or 4 reads in the output (RNA) were excluded from subsequent analysis.
The log(DNA/RNA) ratio (“expression”) was calculated using raw counts, scaled so that the median expression is 0, and the expression levels G+C-content normalized such that the mean expression for every %G+C was 0.
Finally, to eliminate instances where the tag sequence modifies the apparent expression level, any tags containing any one of eight blackballed 5-mer DNA sequences were excluded. Blackballed 5-mers were defined as those for which the absolute value of the average expression level of all tags containing that 5-mer was greater than 0.15.
SNPs were tested for allele-specific reporter activity by a two-sided Student’s t-test, comparing the normalized log(RNA/DNA) expression values for the tags for allele A compared to the tags for allele B. Only SNPs for which we had at least 80 good tags between the two alleles were tested. P-values were corrected for multiple hypothesis testing by Benjamini-Hochberg FDR correction. Only SNPs that had an FDR<0.1 for at least two of the replicates and where the direction of allele- specific reporter activity was consistent between all replicates were considered to be significant.
Genome_build: hg19
Supplementary_files_format_and_content: gzipped tab delimited or fasta; barcodes for each element, enhancer sequences
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| Submission date |
Aug 30, 2019 |
| Last update date |
Apr 01, 2020 |
| Contact name |
Carl G de Boer |
| Organization name |
The Broad Institute
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| Lab |
Aviv Regev
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| Street address |
415 Main St
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE136702 |
Assessing the ability of various genomic features to prioritize causal non-coding variants associated with diseases and traits [MPRA] |
| GSE136703 |
Assessing the ability of various genomic features to prioritize causal non-coding variants associated with diseases and traits |
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| Relations |
| BioSample |
SAMN12668275 |
| SRA |
SRX6785366 |
| 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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