title: Illumina HiSeq 2500 sequencing; RNA-seq of human intestinal organoids infected with Salmonella enterica at 2.5h and 8h post infection
design: RNA-seq of human intestinal organoids infected with Salmonella enterica at 2.5h and 8h post infection
library_construction_protocol: 5 organoids were collected per replicate with 4 replicate for each condition at each time point. HIOs were generated by the In Vivo Animal and Human Studies Core at the University of Michigan Center for Gastrointestinal Research. Human ES cell line WA09 (H9) was obtained from Wicell International Stem Cell Bank and cultured on Matrigel (BD Biosciences)-coated 6-well plates in mTeSR1 medium (Stem Cell Technologies) at 37°C in 5% CO2. Cells were seeded onto Matrigel-coated 24-well plates in fresh mTeSR1 medium and grown until 85 to 90% confluence. Definitive endoderm differentiation was induced by washing the cells with PBS and culturing in endoderm differentiation medium (RPMI 1640, 2% fetal bovine serum [FBS], 2 mM L-glutamine, 100 ng/ml activin A, and 100 U/ml penicillin streptomycin) for 3 days, and media were exchanged each day. Cells were then washed with endoderm differentiation medium without activin A and cultured in mid/hindgut differentiation medium (RPMI 1640, 2% FBS, 2 mM L-glutamine, 500 ng/ml FGF4, 500 ng/ml WNT3A, and 100 U/ml penicillin-streptomycin) for 4 days until spheroids were present. Spheroids were collected, mixed with ice-cold Matrigel (50ml of Matrigel plus 25ml of media plus 50 spheroids), placed in the center of each well of a 24-well plate, and incubated at 37°C for 10 min to allow Matrigel to solidify. Matrigel embedded spheroids were grown in ENR medium (Dulbecco's modified Eagle medium [DMEM]-F12, 1 B27 supplement, 2 mM L-glutamine, 100 ng/ml epidermal growth factor [EGF], 100 ng/ml Noggin, 500 ng/ml Rspondin1, and 15 mM HEPES) for 14 days, and medium was replaced every 4 days. Spheroids growing into organoids (HIOs) were dissociated from Matrigel by pipetting using a cut wide-tip pipette (2 to 3 mm). HIOs were mixed with Matrigel (6 HIOs plus 25ml of media plus 50ml of Matrigel) and placed in the center of each well of 24-well plates and incubated at 37°C for 10 min. HIOs were further grown for 14 days in ENR medium with medium exchanged every 4 days. Before use in experiments, HIOs were carved out of Matrigel, washed with DMEM-F12, and replated with 5 HIO/well in 50ml of Matrigel in ENR medium with medium exchanged every 2 to 3 days for 7 days prior to microinjection. Organoids were microinjected with 10^5 CFU/HIO of each Salmonella strain and samples were harvested at 2.5h and 8h post injection. Transcripts were converted to estimated gene counts using the tximport package with gene annotations from Ensembl. RNA was isolated using the mirVana miRNA isolation kit  Cytosolic and mitochondrial rRNA was removed from samples using the Ribo-Zero gold kit