title: GSM9210464: FACS sorted Venus+ EECs from tHIOs, plate IGB-AA-s026; Homo sapiens; RNA-Seq
library_construction_protocol: Isolation of mouse EECs: Mouse intestinal samples were digested into single cells for 30 to 40 min at 37°C in TrypLETM Express with vigorous up and downs shakings each 5 min. The cells were centrifuged twice at 900rpm, 5min, 4°C in Advanced DMEM, HEPES 10mM, fetal calf serum (FCS) 10%. For the P12 small intestine, an additional filtering step, onto a 70µm cell strainer, was performed before centrifugation. Cells were resuspended in Advanced DMEM F12, HEPES 10mM, FCS 5%, Y-27632 10µM, filtered through a 50µm cell strainer and sorted with a FACS Aria Fusion II (BD) in SORT-seq 384-well cell-capture plates (Single Cell Discoveries). Plates were sealed, centrifuged 2min at 1500g, 4°C, and snap-frozen and stored at -80°C until sequencing. Dissociation of HIOs and tHIOs into single cells and isolation of EECs for single-cell RNA sequencing: Around 40 HIOs (from a single differentiation) were dissociated to single cells as previously described with some modifications (Grün et al., 2015). Briefly, after medium removal, rinsing of each well with PBS1X, and Matrigel disruption, HIOs were dissociated in TrypLE™ Express (1X) (Gibco™) at 37 °C for 20 min, assisted by a mechanical dissociation (up and down with a p1000 pipette). The dissociation was stopped in a basal medium containing Advanced DMEM/F-12, HEPES 10 mM, fetal calf serum 10%, and Y27632 10µM (STEMCELL™ Technologies). After 3 washes, the cells were resuspended in the FACS medium (basal medium containing only 5% of fetal calf serum), strained through a 50 μm cell strainer, and stained with DAPI (to exclude dead cells) before being sorted by flow cytometry (FACSAria Fusion, BD) based on Venus fluorescence levels. Individual cells were sorted directly in pre-filled 384-well cell capture plates (one cell/well) containing the reagents to perform single-cell RNA-sequencing (scRNA-Seq) according to the SORT-Seq technology (Muraro et al. 2016)(Single Cell Discoveries, Utrecht, The Netherlands). tHIOs were dissociated to single cells as described by Yu et al., (2021) with modifications. Briefly, a total of 8 individual tHIOs were harvested 15 weeks post-transplantation. Samples were first minced into small pieces using a scalpel in ice-cold 1X HBSS, then enzymatically digested in the Neural Tissue Dissociation Kit (Miltenyi Biotec, 130-092-628) at 10°C, following the manufacturer's instructions. After complete dissociation, the cells were filtered through a 70µm cell strainer, centrifuged (500g, 5 min, 10°C) and the pellet was resuspended in the basal medium. The cell suspensions (from 1 to 3 tHIOs, depending on their size) were then labeled using an anti-EpCAM antibody to collect only epithelial cells (APC anti-human CD326 (EpCAM) antibody, BioLegend) and DAPI (to exclude dead cells). Cells were filtered through a 50 μm cell strainer before being sorted by FACS (FACSAria Fusion, BD) directly into the 384-well plates for SORT-Seq analysis (Single Cell Discoveries, Utrecht, The Netherlands), as described above. Both DAPI-; EpCAM+; Venus+ or Venus- cells were harvested. Reads were mapped to the human GRCh38 genome assembly. Single cells were sequenced with the SORT-seq method (Muraro et al., 2016), a partially robotized version of the CEL-seq2 protocol (Hashimshony et al., 2016). Library preparation and single-cell RNA sequencing were performed by Single Cell Discoveries (Utrecht, The Netherlands, https://www.scdiscoveries.com). Briefly, single cells were sorted by FACS into each well of a cell-capture 384-well plate, containing well-specific barcoded primers. Each primer contains a cell-specific barcode and a unique molecular identifier (UMI). The protocol involves the following steps: 1) lysis of cells by heat (65°C, 5min) and hybridization of primers to the poly-A tail of mRNAs. 2) Reverse transcription & second-strand synthesis (Invitrogen). 3) Pooling of all the wells. 4) In vitro transcription (IVT) (Ambion) to generate many copies of RNA via linear amplification.5) Fragmentation & reverse transcription: the amplified RNA (aRNA) is fragmented into segments of approximately 300-400 base pairs and reverse transcribed. 6) Amplification of the cDNA by PCR and addition of sequencing adapters (10-12 cycles with Illumina TruSeq primers). The resulting cDNA libraries were paired-end sequenced at a depth of 150.000 reads/cell on an Illumina NextSeq 500.