Single cell RNA sequencing (scRNA-sequencing) from E18.5 wild-type and Tet3 knockout intestinal epithelium
Bibliographic record
Abstract
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 >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).
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.001 | 0.001 |
| Meta-epidemiology (narrow) | 0.002 | 0.001 |
| Meta-epidemiology (broad) | 0.002 | 0.001 |
| Bibliometrics | 0.003 | 0.002 |
| Science and technology studies | 0.002 | 0.001 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.026 | 0.027 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".