|Public on Jun 01, 2020
|Ulcerative Colitis rep 1
|disease: Ulcerative Colitis
|SM buffer or viral-like particle (VLP) preparation from non-IBD or IBD tissue or ileostomy was pre-treated for 30 minutes with polymixin B to ensure endotoxin-free conditions. Media was removed from the cells and SM buffer (for mock condition) or VLP preparation was added drop-wise onto the cells at a volume sufficient to cover the well surface. Treatment was left on cells for 1 hour at 37C with occasional rocking. After 1 hour, treatments were removed entirely, cells were gently washed with PBS, and media was replaced. Cells were collected 24 hours after the end of treatment.
|Peripheral blood mononuclear cells were isolated from the blood of healthy human donors using a Ficoll-based density gradient. Following isolation, adherent cells were cultured on tissue culture-treated plated plates in X-VIVO media containing 100ng/mL rhM-CSF and allowed to differentiate into mature macrophages for 7 days. At this point, cells were counted and replated overnight in fresh media for treatment.
|Cells were lysed in 350µL buffer RLT and RNA was isolated using the Qiagen RNeasy Mini Kit with on-column DNase digest (all according to manufacturer's instruction).
100ng of total RNA from each sample was used for library preparation. First, poly(A)-tailed RNA was enriched for using the NEBnext Poly(A) mRNA magnetic isolation module (E7490; according to manufacturer's instructions). This poly(A)-enriched RNA was then used to construct libraries using the NEBnext Ultra II RNA Library Prep Kit for Illumina (E7770; according to manufacturer's instructions). To allow for multiplexed reads, libraries were dual indexed using indices from NEBnext Multiplex Oligos for Illumina (E6440). Sequencing was done on a NovaSeq S4 flow cell.
|Illumina NovaSeq 6000
|Transcriptome mapping was performed with STAR version 2.5.4a by using the NCBI37 assembly gene annotations
Read counts for individual genes were produced using the unstranded count feature in HTSeq 0.9.1
Supplementary_files_format_and_content: tab-delimited text files include read count for each sample
|Aug 01, 2019
|Last update date
|Jun 02, 2020
|Kate L Jeffrey
|Massachusetts General Hospital
|50 Blossom Street
|Human macrophage responses to viral-like particles from non-IBD or IBD colonic resections