DOI: 10.20350/digitalcsic/17939
title: Single cell RNA sequencing (scRNA-sequencing) from E18.5 wild-type and Tet3 knockout intestinal epithelium

description: Characteristics strain: C57BL6/J genotype: Tet3 knockout tissue: small intestine epithelium Treatment protocol A targeting vector introducing loxP sites around Tet3 exon 5 was constructed and electroporated into (C57BL6/J x C3H/HeJ)F1 embryonic stem cells (ESCs). The construct also contained an FRT-flanked selection cassette coding for neomycin and thymidine kinase. Individual neomycin-resistant ESC clones were screened for recombination in the proper genomic locus. Next, FIAU-negative selection was used to select for clones that had excised the thymidine kinase cassette after transient transfection of an Flp-coding plasmid. Then, a Cre-coding plasmid was transiently transfected to excise the loxP-flanked exon 5 and single ESC clones were genotyped to confirm the excision. Deletion of the floxed region results in a frame-shift that introduces a premature stop codon in exon 6, triggering nonsense-mediated decay of the mutant transcript. Growth protocol Tet3 knockout ESCs were used to generate chimeric mice through aggregation with morula stage (C57BL6/J x C3H/HeJ)F1 embryos. Male chimeric mice were crossed to CD1 females to screen for germ line transmission of the Tet3 knockout allele. Finally, Tet3 heterozygous females were backcrossed with pure C57BL6/JRccHsd males at least 5 generations to obtain an incipient congenic line (N5 or &gt;95% C57BL6/JRccHsd). Animals were housed in a controlled environment with a 12h light/dark cycle in open cages, with access ad libitum to a standard chow diet and water. Mice were time-mated and the presence of a vaginal plug was considered as embryonic day 0.5 (E0.5). All experiments were performed on E18.5 with embryos obtained by caesarian section. All animal experiments were performed in accordance with the animal care guidelines of the European Union (2010/63/EU) prior approval of the Spanish National Research Council Ethics Committee and the Regional Government competent authority. Extracted molecule total RNA Extraction protocol Sorted E18.5 intestinal epithelial cells were fixed in 90% ice-cold methanol and stored at -80ºC until analysis. Tet3 knockout and wild-type samples correspond to a pool of intestinal epithelial cells from two embryos each. For analysis, cells were resuspended in 3x saline sodium citrate buffer containing 0.04% BSA, 1% SUPERase·In RNase Inhibitor (Invitrogen, AM2694) and 40mM DTT (Chen et al, 2018). Single-indexed libraries were prepared using the Chromium Single Cell 3’ v3/v3.1 chemistry (10x Genomics) following the manufacturer’s instructions. 6,000 cells were used per sample. The generated libraries were sequenced on an Illumina Novaseq (Illumina) system using the following transcript read lengths: 28bp for cell barcode and UMI, 8bp for sample index and 91bp for insert. 10,000 reads per cell were sequenced. scRNA-seq analysis was performed at Princess Margaret Genomics Centre (Toronto, Canada).
description: To study the role of tet3 and 5-hydroxymethylation in mouse intestine homeostasis our objectives are: To determine the cell types that show a 5hmC reduction level in the absence of Tet3. Identify the role of Tet3 on transcriptional regulation in the small intestinal epithelium. Define the physiological functions impaired in Tet3 -/- small intestine epithelium.