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| Status |
Public on Oct 02, 2021 |
| Title |
MDBK Cas12/MER41.IFNAR2-/- KO3 BSA rep1 |
| Sample type |
SRA |
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| Source name |
MDBK
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| Organism |
Bos taurus |
| Characteristics |
breed: not applicable
age: not applicable
Sex: not applicable
cell type: MDBK
genotype: enot applicablesCas12a/MER41.IFnot applicableR2-/-
treatment: untreated
timepoint: untreated
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| Treatment protocol |
Cells were either untreated or stimulated with 100 ng/mL bovine IFNG (Kingfisher #RP0013B-005) for 2h or 4h. Monocyte and leukocyte samples were stimulated as PBMC samples, after which Trizol lysates were prepared from the adherent (monocyte) and non-adherent (lymphocyte) fractions.
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| Growth protocol |
MDBK cells were routinely grown at 37C in 5% CO2 and O2 on coated plastic (VWR #10861-680, typically 1,000,000-2,000,000 cells in 10cm dishes) in MEM (Gibco #11095080) supplemented with 1X nonessential amino acids (Gibco #11140050), 1X penicillin-streptomycin (Gibco #15140122), and 10% fetal bovine serum (Gibco #10437010). BL3.1 cells were routinely grown at 37C in 5% CO2 and O2 on uncoated plastic (Greiner Bio-One #82050-856, typically 500,000-1,000,000 cells/mL in T75 flasks) in RPMI 1640 (Gibco #61770036) supplemented with 1X penicillin-streptomycin and 10% fetal bovine serum. Monocytes and leukocytes were derived from PBMC samples from two male “line 999” and are composed of the following breed makeup: 1/2 Angus, 1/4 Gelbvieh, 3/16 Charolais, 1/16 Hereford. Briefly, 15 mL of blood was combined with 15 mL 1X PBS (Gibco #14190144) and 15 mL Histopaque-1077 (Sigma-Aldrich #10771) and centrifuged for 45 minutes at 1,000 x g at room temperature. The monocyte layer was retained, washed in 50 mL of cold 1X PBS, and lysed in 1 mL Red Blood Cell Lysis Buffer (Roche #11814389001) for 5 minutes at 37°C and 5% CO2. The PBMCs were washed three times with 50 mL, 10 mL, and 10 mL cold 1X PBS, respectively, and resuspended in 45 mL of FBS-free RPMI 1640. Monocytes were isolated as described in (Chitko-McKown et al. 2003). Briefly, PBMCs were added to treated T75 flasks and incubated at 37°C and 5% CO2 overnight, and the non-adherent, predominantly leukocyte fraction was removed by washing with PBS. The remaining adherent population represents the monocyte fraction. Leukocytes were derived from the non-adherent fraction. Animals were housed at the U.S. Meat Animal Research Center (USMARC) feedlot in accordance with USDA animal care guidelines.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA from MDBK and BL3.1 was harvested using Zymo Quick-RNA Miniprep Plus Kit (Zymo #R1057). Monocyte and leukocyte lysates were prepared using Trizol (Invitrogen #11596026) reagent, and RNA was extracted using Zymo Direct-zol RNA Miniprep Plus kit (R2071). All lysates were frozen at -80°C prior to extraction, and all extracted RNA were stored at -80°C as single-use aliquots.
Libraries were generated with 200ng or 500ng RNA using KAPA mRNA HyperPrep Kit (KAPA #KK8581) according to the manufacturer's instructions. Briefly, single-indexed adapters (KAPA #KK8700) or unique dual-indexed adapters (KAPA #KK8727) were added to each sample at a final concentration of 78 nM. Excess adapter was removed using sequential 0.63X and 0.70X KAPA Pure Bead (KAPA #KK8002) cleanups. Purified, adapter-ligated samples were amplified for 10 (500ng input RNA) or 12 (200ng input RNA) cycles according to the manufacturer's instructions and purified using a 1.0X KAPA Pure Bead cleanup.
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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 |
Real-Time Analysis v3.4.4 was used for basecalling.
Adapters and low quality reads were trimmed using BBDuk v38.05 with arguments 'ktrim=f k=23 mink=11 hdist=1 maq=10 qtrim=r trimq=10 tpe tbo literal=AAAAAAAAAAAAAAAAAAAAAAA' and mapped to bosTau9 using hisat2 v2.1.0 with options '--rna-strandness RF --no-softclip'. Aligned fragments with an alignment score less than 10 were removed with samtools v1.10. Alignment files corresponding to technical replicates (designated as "s1", "s2", etc.) were merged, and all samples were mapped to genes in the complete refseq annotation using featureCounts v1.6.2 with options '-s 2 -t exon -g gene_id -0 -p'. For visualization aligned fragments were converted to stranded, CPM normalized bigwigs using deepTools bamCoverage v3.0.1.
Differentially expressed genes were called using DESeq2 v1.26.0 with designs '~ treatment` (wildtype) or '~ genotype + treatment + genotype:treatment' (MER41.IFNAR2-/- or MER41.IL2RB-/-). For the mutant differential expression analysis, normalized count data is derived from DESeq2 comparisons between KO IFNG vs WT IFNG. Genes with zero counts across all samples were removed.
Genome_build: bosTau9 (ARS-UCD1.2)
Supplementary_files_format_and_content: Tab-delimited text files with raw fragment counts for each gene
Supplementary_files_format_and_content: CPM normalized signal tracks in UCSC bigwig format
Supplementary_files_format_and_content: Tab-delimited text files including DESeq2 normalized counts
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| Submission date |
Sep 30, 2021 |
| Last update date |
Oct 02, 2021 |
| Contact name |
Edward Chuong |
| E-mail(s) |
edward.chuong@colorado.edu
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| Organization name |
University of Colorado Boulder
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| Department |
BioFrontiers/MCDB
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| Street address |
3415 Colorado Ave, JSC Biotech Bldg
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| City |
Boulder |
| State/province |
CO |
| ZIP/Postal code |
80303 |
| Country |
USA |
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| Platform ID |
GPL26012 |
| Series (2) |
| GSE185081 |
Transcriptional profiling of the innate immune response in bovine cells |
| GSE185082 |
Ruminant-specific retrotransposons shape regulatory evolution of bovine immunity |
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| Relations |
| BioSample |
SAMN21920613 |
| SRA |
SRX12417668 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5605825_MDBK_Cas12_MER41_IFNAR2_KO3_BSA_R1.txt.gz |
139.0 Kb |
(ftp)(http) |
TXT |
| GSM5605825_MDBK_Cas12_MER41_IFNAR2_KO3_BSA_R1_fwd.bw |
51.9 Mb |
(ftp)(http) |
BW |
| GSM5605825_MDBK_Cas12_MER41_IFNAR2_KO3_BSA_R1_rev.bw |
45.0 Mb |
(ftp)(http) |
BW |
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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