GSM6705080: FAC-cyESCs, replicate 3, RNAseq; Macaca fascicularis; RNA-Seq
Freshly collected DC and Si8 from RedMUC298trTg mice were flushed with ice-cold HBSSwo (Mg2+/Ca2+ free supplemented with 10mM HEPES, pH 7.2) to remove luminal content. The tissue was inverted and inflated by injection of HBSSwo. Epithelial isolation was performed by incubation of tissue with pre-digestion buffer (HBSSwo, 5 mM EDTA, 5% v/v FCS, 1 mM DTT) in a shaking incubator. The isolated epithelial cells were resuspended in digestion buffer (HBSS with Mg2+/Ca2+ supplemented with 2 mg/ml collagenase type I and 40 U/ml DNase I) and incubated at 37 °C for 30 min. Cells were washed and re-suspended in PBS and stained with a Viability Dye, washed and resuspended in FACS buffer (HBSSwo, 2% v/v FCS, 5mM EDTA). Alive GCs were sorted using a FACS Jazz (Becton Dickinson) according to the presence or absence of mCherry signal using a C57Bl6 mouse as negative control. Goblet cells were washed in HBSS-/- and resuspended in HBSS-/- supplemented with 0.04% w/v BSA. RNA extraction was performed using a RNeasy Mini Kit (QIAGEN) using DNase treatment (QIAGEN). RNA was analyzed using a 2100 Bioanalyzer (Aglient Technologies). (Transcripts per Kilobase Million). For monkey cells, RNA was extracted with Trizol Reagent (15596026, Invitrogen). The RNA (~50ng) reverse transcription reaction and amplification were performed using SuperScript II (18064-071, Invitrogen), and KAPA HiFi HotStart Ready Mix (KK2602, KAPA). The cDNA was analyzed using a 2100 Bioanalyzer (Aglient Technologies). The RNA Library was generated using TruePrep DNA Library Prep Kit V2 for Illumina (TD501, Vazyme), then the library was adapted for sequencing on an Illmina NovaSeq 6000 platform (sequenced by Annoroad). 