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Status |
Public on May 01, 2024 |
Title |
MPRA, donor7 |
Sample type |
SRA |
|
|
Source name |
Inflammatory (TPP) macrophages
|
Organisms |
Homo sapiens; synthetic construct |
Characteristics |
cell type: Inflammatory (TPP) macrophages donor: donor7
|
Growth protocol |
Monocytes were positively selected using CD14 Microbeads. Macrophage differentiation was performed using conditions that model chronic inflammation (TPP): 3 days GM-CSF (50ng/mL) followed by 3 days GM-CSF, TNFa (50ng/mL), PGE2 (1mg/mL), and Pam3CSK4 (1mg/mL). All cultures were performed at 37C, 5% CO2 in antibiotic-free RPMI1640 media containing 10% FBS, GlutaMAX, and MEM Non-Essential Amino Acids. On day 6 of culture, a minimum of 2x10^7 macrophages were harvested, counted, and nucleofected with the MPRA vector library (aliquots of 5x10^6 cells nucleofected with 5µg vector in 100 uL nucleofection buffer; Human Macrophage Nucleofection Kit, Lonza) using a Nucleofector 2b (Lonza, program Y-011).
|
Extracted molecule |
total RNA |
Extraction protocol |
Macrophages were detached using Accutase and were lysed. RNA was extracted from cell lysates using an AllPrep DNA/RNA Mini Kit (Qiagen). Sequencing libraries were prepared by PCR (30 cycles) using PfuUltra II polymerase (Agilent) and custom primers that annealed to a site within EGFP (F) and the universal primer site in the oligonucleotide (R). Amplified libraries were cleaned using AMPure XP beads (Beckman Coulter): 0.6×, 1.6× and 1.0×. Four sequencing libraries were made from the input MPRA plasmid library (50ng vector, 18 PCR cycles). Libraries were sequenced in pools of 6.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 2500 |
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|
Description |
7TPP
|
Data processing |
Quality control was performed using FastQC (v0.11.9) Barcode counts were generated using a Python script - to be counted a sequenced barcode had to be a perfect match for an oligonucleotide library barcode and be followed by at least 10 bases of the expected constant sequence (XbaI restriction site and GFP). To be deemed a successful transfection, at least 70% of the oligonucleotide library had to be represented in the resulting count file (i.e. count >1). Raw count data was filtered to remove barcodes with a median counts per million (cpm) < 1 in either the mRNA or DNA samples. Barcode counts for identical constructs were collapsed (summed) and quantile normalized (normalize.quantiles function in preprocessCore package in R version 4.1.0) Assembly: hg19 Supplementary files format and content: barcode_counts.txt is a tab-delimited text file containing the raw barcode count data for every sample; normalised_collapsed_constructs.txt is a tab delimited text file containing the filtered, quantile-normalized and collapsed count data for unique genomic constructs tested in the MPRA
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Submission date |
Apr 12, 2023 |
Last update date |
May 01, 2024 |
Contact name |
James C Lee |
E-mail(s) |
james.lee@crick.ac.uk
|
Organization name |
The Francis Crick Institute
|
Lab |
Genetic Mechanisms of Disease
|
Street address |
The Francis Crick Institute, 1 Midland Rd
|
City |
London |
ZIP/Postal code |
NW1 1AT |
Country |
United Kingdom |
|
|
Platform ID |
GPL33331 |
Series (1) |
|
Relations |
SRA |
SRX19940442 |
BioSample |
SAMN34154214 |