GSM7426969: E15.5/16.5 mouse intestine enriched cells; Mus musculus; RNA-Seq
Tissue segments were dissociated to single cells in HBSS buffer containing 10mM HEPES, 1% BSA, 0.3mg/mL DNase and 0.1W/mL Liberase TL on an orbital shaker at 37C for 30 min with trituration with P200 pipette every 15 mins. Single-cell suspensions were then transferred to 96-well U-bottom plates and washed twice in fetal gut media to remove any remaining BSA. MULTIseq lipid barcode labeling was performed in 96 well plates following the MULTI-seq protocol (McGinnis et al., 2019). Once the cells are incubated with lipid modified oligos (barcode, anchor and co-anchor) in total of 100uL volume reaction, 150uL of HBSS with 2% BSA solution were added to quench the reaction and pooled together in 15ml conical tubes. The cells were pelleted and stained with APC/Cy7 anti-mouse Ter119, APC anti-mouse CD31, BV605 anti-mouse CD45, PE/Cy7 Epcam (all from Biolegend) for 30min on ice followed by a wash with FACS buffer. The collected cells were resuspended in FACS buffer with DAPI to stain dead cells and passed through a 35um cell strainer. Using BD FACS Aria II, live (DAPI-, Ter119-) cells as well as enriched rare cells (CD31+, CD45+, Epcam+, or PDGFRa GFP-high) were sorted separately in a desired ratio. 35,000 cells were loaded to each well of GEM chip (2 wells of live-sorted cells and 1 well of enriched rare cells for each experimental day) for single-cell capture using the 10x Chromium Single Cell 3' Reagent Kit V3 (10X Genomics) for aimed target recovery of 20,000 cells per well. Library preparation for both cDNA and MULTI-seq barcodes were performed following 10X library preparation protocol along with MULTI-seq library preparation protocol. Libraries were pooled and subjected to sequencing in a single lane of an Illumina NovaSeq6000 for each experiment.