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| Status |
Public on Sep 30, 2024 |
| Title |
AB34: HEK-293 control 72 h - rep1 |
| Sample type |
SRA |
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| Source name |
HEK-293 control 72 h - rep1
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK-293
cell type: human embryonic kidney cells
treatment: unexposed
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| Treatment protocol |
Adherent human cells were exposed to Borrelia burgdorferi strain B31 for 72 h in antibiotic free media. The multiplicity of infection (MOI) was 50:1 bacteria cells: human cells.
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| Growth protocol |
All human cells were grown at 37°C with 5% CO2. HUVECs required optimized endothelial cell growth media EGMTM BulletKitTM (Lonza, CC-3124). HEK-293 cells were grown in DMEM media (Lonza, 12-733F) supplemented with 10% v/v heat-inactivated FBS (Rockland, FBS.02-0500), each 1% v/v L-glutamine (Lonza, 17-605E), sodium pyruvate (Lonza, 13-115E), and penicillin/streptomycin (Sigma-Aldrich, P4333). Borrelia burgdorferi strain B31 was cultured in Barbour-Stoenner-Kelly medium including 6% rabbit serum (BSK-H, Darlynn biologicals, BB83-500), supplemented with antibiotics (Sigma-Aldrich) at the final concentration of 50 µg/ml Rifampicin (R3501), 20 µg/ml Phosphomycin (P5396), and 2.5 µg/ml Amphotericin B (A9528) in glass culture tubes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extraction was performed with the RNeasy Mini Kit (74104, Qiagen) including DNase I on-column digestion (1010395, Qiagen) and elution in 50 µl RNase-free water.
The library for RNA-seq was prepared with 200 ng RNA input and the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs (NEB), E7760L). First, mRNA was enriched using the NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490L) and following the manufacturer’s instructions. Briefly, after isolation, samples were fragmented for 15 min at 94°C for a target insert size of 200 bp. Next, the first and the second cDNA strand were synthetized, followed by an Ampure XP bead (Beckman Coulter, A63881) clean up. Then the samples were end-prepped and the adaptor was ligated. The adaptor was diluted following the manufacturer’s instructions. The adaptor-ligated samples were purified with Ampure XP beads before amplification. A different Unique Dual Index Primer Pair (NEB, E6440S) was added to each sample and 11 cycles were used for the PCR enrichment. Purification using Ampure XP beads was used to obtain the final library. The quality and quantity of the library was assessed with the D1000 assay on the Tapestation (Agilent, 5067-5582 and 5067-5583) and the dsDNA HS assay from Qubit (Thermo Fisher, Life Technologies, Q32851).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
adapter trimming with BaseSpace™ Sequence Hub Prep platform was followed by trimming with Trim Galore (v.0.4.4, Krueger, 2016) with default settings.
pseudo alignment and quantification was performed with Salmon (v.1.4.0, Patro et al., 2017) in reference to the human transcriptome from Gencode (Release 37 - GRCh38.p13).
Low-expressed genes were filtered out, and only genes with at least more than 1 read in at least 2 samples in one of the groups were considered for further analysis.
normalization and differential expression of the count matrix was performed with DESeq2 (v.1.28.1, Love et al., 2014).
Genome_build: hg19
Supplementary_files_format_and_content: HUVEC_rawcounts.csv: Matrix table with raw counts for every gene and every HUVEC sample
Supplementary_files_format_and_content: HEK-293_rawcounts.csv: Matrix table with raw counts for every gene and every HEK-293 sample
Supplementary_files_format_and_content: BOTH_rawcounts.csv: Matrix table with raw counts for every gene and every cell type HUVEC and HEK-293
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| Submission date |
Jan 24, 2022 |
| Last update date |
Sep 30, 2024 |
| Contact name |
Anne Berthold |
| Organization name |
Mount Allison University
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| Department |
Biology
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| Lab |
Lloyd tick lab
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| Street address |
62 York Street
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| City |
Sackville |
| State/province |
New Brunswick |
| ZIP/Postal code |
E4L 1E2 |
| Country |
Canada |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE194294 |
Genome-wide transcriptome analysis of human cell models exposed to Borrelia burgdorferi |
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| Relations |
| BioSample |
SAMN25228493 |
| SRA |
SRX13885801 |
| 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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