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| Status |
Public on May 15, 2023 |
| Title |
IgE+Monocytes, Allergic, Clinical Phase EL16 |
| Sample type |
SRA |
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| Source name |
IgE-binding Monocyte
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| Organism |
Equus caballus |
| Characteristics |
cell source: Heparinized Blood
cell type: IgE-binding Monocyte
allergy status: Allergic
disease stage: Clinical Phase
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Frozen IgE-binding monocytes were thawed at 37ºC and slowly resuspended into 10ml PBS-FCS (PBS supplemented with 20% FCS and 2mM EDTA (Boston BioProducts, Boston, NY, USA) at RT. Cells were pelleted at 100xg, 5 min, RT and washed once in 1ml PBS-FCS. Cells were resuspended in 250µl PBS-FCS and 750µl Trizol LS solution (Invitrogen, Waltham, MA, USA). Then, 200µl chloroform (Fisher Scientific, Pittsburgh, PA, USA) was added. Tubes were vigorously shaken for 15 seconds and centrifuged at 16000xg, 15 min, 4ºC. The aqueous phase was collected and added to an additional 600µl chloroform in Phase Lock Gel tubes (Quantabio, Beverly, MA, USA). To improve RNA recovery, 2µl of a glycogen carrier (GlycoBlue, ThermoFisher Scientific, Waltham, MA, USA) was added. Last, 500µl isopropanol was added and incubated on ice for 1 hour, then washed three times in 75% ice-cold ethanol. All wash spins were performed at 16000xg, 10 min, 4ºC. Extracted RNA was air dried in a laminar flow hood at RT, then resuspended in 20µl RNase-free water (Invitrogen, Waltham, MA, USA), and stored at -80ºC until submitted for sequencing.
RNA was first poly-A selected with the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA) and TruSeq-barcoded RNA-seq libraries were generated using the NEBNext® Ultra™ II Directional RNA Library Prep kit (New England Biolabs, Ipswich, MA, USA). Libraries were sequenced on an Illumina instrument with a read length of 75bp single end reads at a depth of 20 million reads per sample.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
IgE+Monocytes, Allergic, Clinical Phase
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| Data processing |
Raw sequencing reads were initially processed by Cornell’s TREx Facility using pipelines built around ENCODE standards and practices. First, raw fastq files were processed with TrimGalore v0.6.0 to remove low quality bases and adaptor sequences.
Reads were then mapped to the EquCab3.0 genome using STAR v2.7.0e.
Read count data was further analyzed using iDEP.96 (integrated Differential Expression and Pathway analysis). Counts data was transformed using EdgeR (log2(CPM+4)) on all genes with a minimum of 0.5 counts per million (CPM) in at least 1 library. Samples from each phase were normalized separately.
Assembly: EquCab3.0 genome
Supplementary files format and content: tab-delimited text files include counts per million (CPM) values for each Sample
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| Submission date |
May 01, 2023 |
| Last update date |
May 15, 2023 |
| Contact name |
Bettina Wagner |
| E-mail(s) |
bw73@cornell.edu
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| Organization name |
Cornell University
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| Department |
Population Medicine and Diagnostic Sciences
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| Street address |
240 Farrier Drive
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| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14815 |
| Country |
USA |
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| Platform ID |
GPL21401 |
| Series (1) |
| GSE231427 |
IgE-binding monocytes upregulate the coagulation cascade in allergic horses |
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| Relations |
| BioSample |
SAMN34507140 |
| SRA |
SRX20177558 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7277556_A2-T2_rawCounts.txt.gz |
108.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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