GSM9743739: control_4h_2; Homo sapiens; RNA-Seq
Monolayers were cultured on 12 mm diameter 0.4 um polycarbonate filters (Millicell PIHP01250) and differentiated as described above. Spores were treated with glycine and sodium cholate, as above, collected by centrifugation (Eppendorf 5415, 13200 rpm, 5 min) resuspended in PBS and 1 x 105 spores in 10 ul or PBS was added to the apical medium. Transwells were incubated as described above. After 4, 15, 20 or 25 hours of incubation, monolayers were washed with 100 ul PBS, EDTA 0.5 mM, pH 8 and incubated with TrypLE Express, 600 ul in the basolateral and 400 ul in the apical compartments at 37oC for 12 min. One hundred ul 50% FBS was added to the apical compartment and the cells were suspended by pipeting up and down several times. Cells were passed through a 40 um cell strainer and recovered in tubes precoated with 0.1% BSA. The transwells were then washed with 400 ul CMGF-, which was passed through the strainer and combined with the previous filtrate. Cells were collected by centrifugation at 300g x 5 min. Most of the supernatant was gently removed. Cells were resuspended in 1 ml of 0.04% BSA, 5 uM Y27632 and collected as above. Using a wide bore tip, the pellet was resuspended in the remaining supernatant by pipetting up and down. Cells were counted with a Corning Cytosmart counter and adjusted to 1 x 106 cells/ml. The remaining steps were performed following manufacturer's protocol. Briefly, the cell suspension was loaded along with reagents, barcoded Single Cell 3' v3.1 Gel Beads and Partitioning Oil onto the Chromium Next GEM Chip G. Cells were partitioned into GEMS using the Chromium Controller. Gel beads were then dissolved, releasing primers, and the cells were lysed. Reverse transcription was performed to generate cDNA tagged with cell specific barcodes and Illumina TruSeq Read 1 sequencing primer. GEMS were broken and first strand cDNA was purified and PCR amplified. After enzymatic fragmentation followed by size selection, the cDNA was PCR amplified, incorporating a sample index and primers P5, P7, and TruSeq Read 2 for Illumina sequencing. Library quality was assessed using an Agilent Bioanalyzer. Paired end sequencing was performed by the Tufts University Genomics Core Facility.