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Sample GSM7359867

Query DataSets for GSM7359867

Status

Public on Sep 12, 2023

Title

I2_1

Sample type

SRA

Source name

Ileum

Organism

Sus scrofa

Characteristics

tissue: Ileum
tissue dissection: Distal ileum with Peyer's patches
breed: Mixed-breed
age: 8 weeks
gender: Female

Extracted molecule

polyA RNA

Extraction protocol

Single-cell RNA sequencing: Reagents were equilibrated and processes carried out at room temperature (RT) unless stated otherwise. ~2-inch-length tissue cross-sections were placed into 30 mL of 2 mM ethylenediaminetetraacetic acid (EDTA; Invitrogen AM9260G), 2 mM L-glutamine (Gibco 25-030), 0.5% bovine serum albumin (BSA; Sigma-Aldrich A9418) in Hank’s balanced salt solution (HBSS; Gibco 14175) for transport. Tissues were cut open, rinsed with phosphate-buffered saline (PBS), pH 7.2 (made in-house), the muscularis layer was removed, and ~1.5 g of tissue was incubated in 30 mL of 5 mM dithiothretol (Invitrogen 15508), 2% fetal calf serum (FCS; heat-inactivated; Gibco A38401) in HBSS for 20 min, 37C, 200 rpm. Tissues were washed in 20 mL of 10 mM HEPES (Fisher BioReagents BP299) in HBSS for 10 min, 37C, 200 rpm, followed by transfer into a C-tube (Miltenyi 130-093-237) containing 15 mL of 10 mM HEPES, 0.2U/mL Liberase TM (Roche 5401127001), 30 ug/mL DNase I (Sigma D5025) in HBSS. Mechanical dissociation was executed with a gentleMACs Octo Dissociator (Miltenyi 130-095-937) intestine C-tube protocol. C-tubes were incubated 45 min, 37C, 200 rpm, followed by another round of mechanical dissociation. 10 mL of 2 mM EDTA, 2 mM L-glutamine, 0.5% BSA in HBSS was added to C-tubes, and samples were filtered sequentially through non-woven surgical gauze sponges (Starryshine GZNW44), 100-micron nylon filters, and 40-micron nylon filters. Collected suspensions were centrifuged 5 min, 200 xg and resuspended in 2 mM L-glutamine, 2% FCS in HBSS. Density gradient centrifugation was performed to enrich for lymphocytes by overlaying cell suspensions onto Histopaque 1077 (Sigma-Aldrich) and centrifuging 30 min, 400 xg, 22C. Cell fractions were collected from the interphase layer and washed with 2 mM L-glutamine, 2% FCS in HBSS. Final cell suspensions were poured over 40-micron nylon filters.
Tissues were embedded in optimal cutting temperature (OCT) compound (Sakura 4583) and snap frozen in an isopentane bath cooled with liquid nitrogen. Tissues were stored at -80C. For cryosectioning, tissue blocks were equilibrated to -20C for 20 minutes. On a cryostat (Leica CM 1860), OCT blocks were cut and faced to an appropriate plane and size that included transverse profiles of Peyer’s patches, lamina propria, and epithelium. 10-micron tissue sections were adhered to VIsium slides (10X Genomics).
Single-cell RNA sequencing: A target of 10,000 isolated cells per sample were partitioned using the Chromium Single Cell 3’ v3 Chemistry (10X Genomics CG000183). Samples were sequenced using 2x100, paired-end sequencing on a HiSeq 3000 and NovaSeq SP flow cell (Illumina).
Spatial transcriptomics: Using optimized permeabilization parameters, libraries were created using the Visium Spatial Gene Expression Reagent Kit (10X Genomics PN-1000186), Visium Spatial Gene Expression Slide Kit (10X Genomics PN-1000185), and Library Construction Kit (10X Genomics PN-1000190) according to procedures in User Manual CG000239 (10X Genomics). Tissue bluing was omitted from the manufacturer’s protocol. Tissue images were digitally scanned without coverslips by an Aperio AT2 whole slide imager (Leica Biosystems) at 40X (0.25 um/pixel) magnification. Samples were sequenced on a NovaSeq SP flow cell (Illumina) with 2x100, paired-end sequencing.

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

I2_S1
scRNA-seq of Sus scrofa: 8 weeks old female distal ileum with Peyer's patches

Data processing

Read were aligned using Cell Ranger v6.1.2 (single-cell) and Space Ranger v1.3.0 (spatial)
Assembly: Sus Scrofa 11.1
Annotation build: Sus Scrofa 11.1.97

Submission date

May 16, 2023

Last update date

Sep 12, 2023

Contact name

Sathesh K Sivasankaran

E-mail(s)

sathesh@iastate.edu

Organization name

University of Missouri

Lab

Bioinformatics and Analytics Core