GSM7432694: ZO1_capillary_3; Homo sapiens; RNA-Seq
differentiation protocols: iECs and iBMECs were differentiation from the WTC-ZO1 iPSC line (AICS-0023 cl.20) following previously published protocols (see PMID: 36333694).  Standard culture mediums were used for the duration of experiments. lentivirus production: For each transcription factor, a Gateway-compatible pShuttle vector containing untagged an ORF and a stop codon were obtained and inserted into a doxycycline-inducible backbone pLIX401 (Addgene # 41393) following Genecopoeia protocols.  A third-generation lentivirus system was employed using plasmids pRSV-Rev (Addgene #12253), pMDLg/pRRE (Addgene #12251), and pMD2.G (Addgene #12259).  A Lenti-X™ qRT-PCR Titration Kit (Takara #631235) was used to calculate physical titer following manufacturer guidelines, with functional titer determined using a control virus expression EYFP. transduction protocol (iEC only): ~3350 iECs were plated into each well of a 96-well plate and immediately exposed to lentivirus particles at an MOI of ~3-5 (based on functional titer estimated using pLIX401.EYFP virus. Transduction efficiency was improved by using 10 ug/mL polybrene for 1 hour at RT at 800g in a swinging bucket centrifuge (spinfection), control iECs were not transduced but still exposed to polybrene. transduction protocol: After 1 hour spinfection, media was replenished and cells were cultured for 48 hours. Then transduced cells were selected (0.5 ug/mL puromycin) for 3 days. Then expression of transgene was induced (10 ug/mL doxycycline) for 3 days. Monolayers on 96 well plates were washed with 1xPBS, treated with 0.25% trypsin-EDTA for 10 minutes, and then spun down in a V-bottom 96 well plate (x3) to thoroughly wash. SMART-Seq mRNA LP kit (Takara Bio #634769) was used according to manufacturer protocols.