title: HT135 Intestine CD31+CD144+
organism: Homo sapiens
description: Protocols: De-identified human fetal lung, intestine, and kidney tissue was obtained from the University of Washington Laboratory of Developmental Biology. Tissue was shipped overnight in Belzer-UW Cold Storage Solution (ThermoFisher, NC0952695) with cold packs (Menon et al., 2018; Miller et al., 2020). Primary human tissue was dissociated into single cell suspensions for FACS isolation of ECs according to a previously published protocol (van Beijnum et al., 2008). Single cell suspensions were passed through a 70µm filter, pelleted, and resuspended in staining buffer comprised of PBS with 1x PenStrep, 2 µM EDTA, and 2% FBS. Cells were stained with CD144-APC and CD31-PE or corresponding isotype controls for 30 minutes on ice in a total volume of 100 µl per 1x107 cells. Cells were washed three times in excess staining buffer accompanied by centrifugation at 300xg for 5 mins between washes. Cells were resuspended in staining buffer with DAPI (0.2 µg/ml). Antibody stained samples and controls were analyzed and sorted on a FACSAria III cell sorter (BD), and analysis was performed using BD FACSDiva software. Any post-acquisition analysis was performed using FlowJo. Cells were sorted into staining buffer, and after sorting cells were snap frozen and stored at -80 oC prior to RNA isolation. RNA was isolated from snap frozen cell pellets using the RNeasy Mirco Kit (74004, Qiagen), according to manufacturer's guidelines. cDNA libraries were prepared using the SMARTer Stranded Total RNA-Seq Kit v2- Pico Input (634412, Takara) by the University of Michigan Advanced Genomics Core according to manufacturer's guidelines.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-12-20T04:16:53Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-07-17T11:43:25Z
attributes.tag: External Id
attributes.value: SAMEA7066482
attributes.tag: INSDC center name
attributes.value: University of Michigan Health System - Internal Medicine - Gastroenterology
attributes.tag: INSDC first public
attributes.value: 2020-12-20T04:16:53Z
attributes.tag: INSDC last update
attributes.value: 2020-07-17T11:43:25Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9372:HT135 Intestine CD31+CD144+
attributes.tag: age
attributes.value: 14
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: gut endothelial cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: late embryonic stage
attributes.tag: disease
attributes.value: normal
attributes.tag: individual
attributes.value: 10
attributes.tag: organism part
attributes.value: intestine
attributes.tag: phenotype
attributes.value: CD31+CD144+
attributes.tag: sample name
attributes.value: E-MTAB-9372:HT135 Intestine CD31+CD144+
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: sex
attributes.value: female