|
| Status |
Public on May 01, 2024 |
| Title |
MPRA, donor6 |
| Sample type |
SRA |
| |
|
| Source name |
Inflammatory (TPP) macrophages
|
| Organisms |
Homo sapiens; synthetic construct |
| Characteristics |
cell type: Inflammatory (TPP) macrophages
donor: donor6
|
| 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.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
6TPP
|
| 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
|
| |
|
| 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 |
SRX19940441 |
| BioSample |
SAMN34154215 |
