GSM6586007: colon epithelium from ILC2-deficient animals, N.b. infected; Mus musculus; RNA-Seq
The intestine was incubated in PBS supplemented with 30 mM EDTA (Roboklon) and 1.5 mM DTT (Sigma-Aldrich) for 20 min on ice. Next, the tissue was transferred to prewarmed PBS/30 mM EDTA and incubated on a shaker at 37°C for 10 minutes and vortexed afterwards to remove the epithelium. The epithelium was washed in PBS/10% FCS and afterwards incubated in HBSS (Sigma-Aldrich) supplemented with dispase (0.5 U/ml; Corning) on a shaker at 37°C for 10 minutes and another 5 minutes after addition of FCS (1% final concentration) and DNaseI (100 μg/ml; Sigma-Aldrich) and filtered through a 70 µm and a 40 µm cell strainer. The purified cDNA was amplified using 11 cycles of PCR and then used for 3' gene expression (GEX) library preparation. Through fragmentation, adapter ligation and index PCR the final libraries were obtained. The quality of single cell 3' GEX was assessed by Qubit quantification and Tapestation analysis (HS D1000, Agilent). The sequencing was performed on a NovaSeq6000 (Illumina) with a S1 xp flow cell, paired-end sequencing with 400mln reds per library and otherwise the recommended sequencing conditions for 3' GEX (read1: 28nt, read2: 90nt, index i7: 10nt, index i5: 10nt).