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| Status |
Public on Jan 31, 2021 |
| Title |
SLE_Jurkat_RNA_TNFa_4 |
| Sample type |
SRA |
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| Source name |
Jurkat cell line
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| Organism |
Homo sapiens |
| Characteristics |
replicate: 4
cell line: Jurkat
disease state: Systemic Lupus Erythematosus (SLE)
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| Treatment protocol |
For each experimental group, we collected a total of 5 x 10E7 cells per replicate for 5 replicates. Cells were then suspended with 50 g transfection library plasmid in 400 L Buffer R. Electroporation was performed with the Neon transfection system in 100l needles with 3 pulses of 1350V, 10 ms each. After transfection, cells were recovered in 50 mL pre-warmed RPMI medium supplemented only with 10% FBS for 24 hrs. After recovery, cells were supplemented with or without 100ng/ml TNF for 24 hrs. Cells were then collected for preparation of the sequencing library for barcode counting.
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| Growth protocol |
Jurkat cell line was grown in RPMI medium supplemented with 10% FBS, 100 units/mL of penicillin, and 100 µg/mL of streptomycin. Cells were seeded at a density of 5 x 10E5 cells/mL the day before transfection.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total DNA and RNA of transfected cells were extracted by the Qiagen ALLPrep DNA/RNA Mini Kit following the manufacturer’s instruction
Extracted RNA was subjected to DNase treatment in a 375 L system with 2.5 L Turbo DNase, and 37.5 L Turbo DNase Buffer at 37°C for 1 hr. 3.75 L 10% SDS and 37.5 L 0.5M EDTA was added to stop DNase with 5 mins of incubation at 75°C. The whole volume was used for eGFP probe hybridization in an 1800 L system, with 450 l 20X SSC Buffer, 900 L Formamide, and 1 L of each 100 M Biotin-labeled GFP probe One to Three. The probe hybridization was performed through incubation at 65°C for 2.5 hrs. 200 l Dynabeads™ MyOne™ Streptavidin C1 was prepared according to the manufacturer’s instructions. The beads were suspended in 250 L 20X SSC Buffer and incubated with the above probe hybridization reaction at room temperature for 15 mins. Beads were then collected on a magnet and washed with 1X SSC Buffer once, and 0.1X SSC Buffer twice. eGFP mRNA was eluted first through adding 12.5 L ddH2O, heating at 70°C for 2 mins and collecting on a magnet, then adding another 12.5 L ddH2O, heating at 80°C for 2 mins and collecting on a magnet. All collected elution was performed with another DNase treatment in a 30 L system containing 0.5 L Turbo DNase, and 3 L Turbo DNase Buffer at 37°C for 1 hr. 0.5 L 10% SDS was added to halt DNase reactions. Eluted mRNA was purified through RNA Clean SPRI Beads. mRNA was reverse transcribed to cDNA using SuperScript™ IV First-Strand Synthesis System with specific primer MPRA_v3_Amp2Sc_R, following the manufacturer’s instructions. cDNA, extracted DNA and plasmid control were then used for building sequencing libraries following Tag-seq Library Construction section in the paper of Tewhey et al (PMID: 27259153). A total of two PCR rounds were needed for building the sequencing library. The first PCR was performed with TruSeq_TruSeq_Universal_Adapter_P5 and MPRA_V3_Illumina_GFP_F. The second PCR was performed with TruSeq_TruSeq_Universal_Adapter_P5 and index primer. Samples were purified, molar pooled and sequenced using Illumina 1x100bp on Illumina Novaseq 6000
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: Massively parallel reporter assay (MPRA)
Paired-end, 125 bp reads were first quality filtered through Trimmomatic-0.3864 (flags: PE -phred33, LEADING:25, TRAILING:25, MINLEN:80). Read 1 was then separated into the 20bp barcode region and the oligo-matching region. The trimmed oligo-matching region of Read 1 and Read 2 were mapped back to the synthesized oligo sequences using Bowtie265 (flags: -X 250, --very-sensitive, -p 16). Barcodes were then associated with the oligo sequences using the read ID. Only uniquely mapped barcodes were used for downstream analysis.
Single end reads were first quality filtered using Trimmomatic-0.3864 (flags: PE -phred33, LEADING:3, TRAILING:3, MINLEN:70). Each read was then separated into the 20bp barcode region and the constant region. The trimmed constant regions of the reads were mapped back to the constant regions within the GFP 3’ UTR using Bowtie265 (flags: --very-sensitive, -p 16). Only reads with Levenshtein distance of 4 or less within the constant region and perfect matches to the two bases directly adjacent to the barcode were kept. Barcodes were then associated with the retained reads using the read ID. Only barcodes that met our quality threshold requirements were used for downstream analysis.
We followed the procedures described in the “Identification of Regulatory Oligos” section of Tewhey et al (PMID: 27259153) with minor modifications. In brief, oligos (alleles) with 30 or more unique barcodes from the plasmid control were included for analysis. Barcode count totals for each oligo, including SLE variants and the 20 control variants, were passed into DESeq2 (R version 3.5.367) to estimate the fold change and significance between plasmid controls and the triplicate experiments. An adjusted p-value of less than 0.05 was required for significance. Only significant alleles with greater than or equal to a 1.5x fold change were identified as enhancer alleles (enAlleles). A variant was identified as an enhancer variant (enVar) if any allele of this variant was an enAllele. Results for the 20 control variants were compared to data from Tewhey et al (PMID: 27259153) to estimate accuracy, sensitivity, and specificity.
Only enVars were considered for allelic analysis. The barcode counts from every allele of enVars were used for calculating p-values by comparing the log2 ratios of the non-reference allele vs the reference allele, normalized by plasmid controls, using Student’s t-test (PMID: 27259153). P-values were adjusted with the Benjamini-Hochberg FDR-based procedure. A corrected p-value of less than 0.05 was required for significance. Only significant alleles with 25%-fold changes or greater were identified as allelic enVars. We have created an R package (mpraprofiler) for performing this analysis, which is available on the Weirauch lab GitHub page (https://github.com/WeirauchLab/).
Genome_build: hg19
Supplementary_files_format_and_content: tab delimited file: barcodes and count for each element, enhancer sequences. xlsx: summarized counts from all data
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| Submission date |
Nov 17, 2020 |
| Last update date |
Jan 31, 2021 |
| Contact name |
Xiaoming Lu |
| Organization name |
Cincinnati Children's Hospital Medical Center
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| Department |
Center for Autoimmune Genomics and Etiology (CAGE)
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| Lab |
Leah Kottyan
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| Street address |
3333 Burnet Ave
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| City |
Cincinnati |
| State/province |
Ohio |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE143792 |
Global discovery of SLE genetic risk variant allelic enhancer activity |
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| Relations |
| BioSample |
SAMN16817002 |
| SRA |
SRX9521014 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4911336_Jurkat_TNFa_RNA_4_barcode_count.txt.gz |
30.8 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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