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Sample GSM7277552 Query DataSets for GSM7277552
Status Public on May 15, 2023
Title IgE+Monocytes, Healthy, Remission Phase EL11
Sample type SRA
Source name IgE-binding Monocyte
Organism Equus caballus
Characteristics cell source: Heparinized Blood
cell type: IgE-binding Monocyte
allergy status: Healthy
disease stage: Remission Phase
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.
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
Description IgE+Monocytes, Healthy, Remission Phase
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
Submission date May 01, 2023
Last update date May 15, 2023
Contact name Bettina Wagner
Organization name Cornell University
Department Population Medicine and Diagnostic Sciences
Street address 240 Farrier Drive
City Ithaca
State/province NY
ZIP/Postal code 14815
Country USA
Platform ID GPL21401
Series (1)
GSE231427 IgE-binding monocytes upregulate the coagulation cascade in allergic horses
BioSample SAMN34507144
SRA SRX20177554

Supplementary file Size Download File type/resource
GSM7277552_H4-T1_rawCounts.txt.gz 112.4 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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