Customer Developed Protocol

Generation of single cell suspension from E8.25mouse embryos
Contributed by:

Bertie Gottgens, Ph.D.

CUSTOMER DEVELOPED PROTOCOL
Generation of single cell suspension from E8.25 mouse embryos

Contributor Research Profile
The Göttgens group uses a combination of experimental and computational approaches to study how transcription
factor networks control the function of blood stem cells and how mutations that perturb such networks cause
leukaemia. This integrated approach has resulted in the discovery of new combinatorial interactions between keyblood stem cell regulators, as well as experimentally validated computational models for blood stem cells. Current
research focuses on (i) single cell genomics of early blood development, (ii) computer models to chart thetranscriptional landscape of blood stem and progenitor cell differentiation, (iii) transcriptional consequences ofleukaemogenic mutations in leukaemia stem/progenitor cells, and (iv) molecular characterization of human bloodstem/progenitor cell populations used in cell and gene therapy protocols.
Link to the lab: https://www.stemcells.cam.ac.uk/research/pis/gottgens

Reference
This protocol was used in:
Ibarra-Soria et al. Defining murine organogenesis at single-cell resolution reveals a role for theleukotriene pathway in regulating blood progenitor formation., Nat Cell Biol. 2018 Feb;20(2):127-134
https://www.ncbi.nlm.nih.gov/pubmed/29311656
Publication Press Release: https://www.stemcells.cam.ac.uk/news/new-2018cell-map2019-reveals-intricatedetails-of-mammalian-development

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
Generation of single cell suspension from E8.25 mouse embryos

Required Buffers and Reagents
1. PBS with 3% FBS
2. TrypLE
3. 0.04% BSA
4. Flowmi Cell Strainer (40 micrometer)ProtocolUteri were harvested from time-mated dams and embryos micro-dissected in ice-cold 3% FBS in PBS. Embryos
were pooled together by developmental stage and larger later stage embryos fragmented using dissecting
forceps for more efficient dissociation. Embryo fragments were transferred to ice-cold 3% FBS/PBS in 1.5mL
Eppendorf tubes. The embryo fragments were centrifuged at 400G for 5 minutes, the supernatant aspirated andre-suspended in 50-100 µL of TrypLE ensuring samples were adequately submerged. Samples were incubated at37°C in a shaker at 1400rpm for 2-5 minutes depending on developmental stage. The resulting suspensions werevisually inspected to ensure adequate dissociation prior to quenching with FBS and wash (400G, 5 minutes) with
3% FBS/PBS. Cell suspensions were centrifuged and resuspended in 70 - 100 µL of 0.04% BSA. Intermediate cellcounts were taken at the quenching stage and in combination with target cell recovery used to inform final
resuspension volume. The cells suspended in 0.04% BSA were filtered through a 40 micrometer Flowmi cellstrainer into a new 1.5mL Eppendorf and the final cell count performed.

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