Customer Developed Protocol

Cell Dissociation and Crypt Isolation of theMouse Small Intestine
Contributed by:Aviv Regev, Regev Lab, Broad Institute

CUSTOMER DEVELOPED PROTOCOL
Cell Dissociation and Crypt Isolation of the Mouse Small Intestine

Contributor Research Profile
The Regev laboratory studies biological circuits, gene regulation, and evolution. Lab members pursue multidisciplinary projects that aim to determine how complex molecular circuits function and evolve in response togenetic and environmental changes, cellular differentiation, evolution, and disease. Aviv Regev and her colleagues
develop experimental and computational approaches to systematically decipher the mechanisms that underlie thetranscriptional regulatory circuits in organisms ranging from yeast to humans.
Link to the lab: https://www.broadinstitute.org/regev-lab

Reference
This protocol was used in:
Haber et al. A single-cell survey of the small intestinal epithelium, Nature, 2017, 551(7680):333-39
https://www.ncbi.nlm.nih.gov/pubmed/29144463

10x Genomics Products
10x Chromium™ Single Cell Gene Expression Solution - https://www.10xgenomics.com/single-cell/

Notices
THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis Customer Developed Protocol is provided for general information only and is not directly supported,
endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes no claims about theprovided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

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CUSTOMER DEVELOPED PROTOCOL
Cell Dissociation and Crypt Isolation of the Mouse Small Intestine

Required Buffers and Reagents
1. PBS (cold)
2. 20 mM EDTA-PBS
3. TrypLE express (Invitrogen)4. 40-μm filter
5. 0.4% BSA–PBS
6. FACS machine and cell stain

ProtocolCell dissociation and crypt isolation
The small intestine of C57BL/6J wild-type, Lgr5–GFP or Gfi1b–GFP mice was isolated and rinsed in cold PBS. Thetissue was opened longitudinally and sliced into small fragments roughly 2 mm in length. The tissue wasincubated in 20 mM EDTA–PBS on ice for 90 min, shaking every 30 min. The tissue was then shaken vigorously
and the supernatant was collected as fraction 1 in a new conical tube. The tissue was incubated in fresh EDTA–
PBS and a new fraction was collected every 30 min. Fractions were collected until the supernatant consisted
almost entirely of crypts. The final fraction (enriched for crypts) was washed twice in PBS, centrifuged at 300g
for 3 min, and dissociated with TrypLE express (Invitrogen) for 1 min at 37 °C. The single-cell suspension wasthen passed through a 40-μm filter and stained for FACS for scRNA-seq (below) or used for organoid culture. We
confirmed the robustness of this method by testing additional single-cell isolation methods—either ‘whole’(scraping the epithelial lining) or ‘villus-enriched’ (fraction 1; see above)—and found that, owing to the highmortality rate (via anoikis) of post-mitotic differentiated cells (the primary component of which is matureenterocytes), crypt-enriched single-cell suspension faithfully represents the composition of the types of small
intestine cell (data not shown).

Cell Sorting
A FACS machine (Astrios) was used to sort single cells into an Eppendorf tube containing 50 μlof 0.4% BSA–PBS and stored on ice until proceeding to the GemCode single-cell platform.

THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis 10x Community Customer Developed Protocol is provided for general information only andis not directly supported, endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes
no claims about the provided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM