Selected oocytes or embryos were transferred to acidic Tyrode’s solution (pH 2.5, Sigma) to remove the zona pellucida. Zona-free embryos were then incubated for 10 min (for the 2-, 4-, and 8-cell stage embryos) or 20 min (for morulae) in Accutase medium (Gibco) before dissociation into single embryo cells by careful pipetting. We then manually separated blastocysts into ICM and TE using laser microdissection. ICM and TE compartments were subsequently placed into Accutase medium for 30 min and washed thoroughly in phosphate-buffered saline (PBS) with 0.5% (m/v) bovine serum albumin (BSA) (Sigma). We isolated single embryo cells by gentle, repeated pipetting; washed them 3–5 times in PBS with 0.5% BSA; and placed the cells into a PCR tube for LiCAT-seq library preparation.
Accutase medium
Manufactured by Thermo Fisher Scientific
Accutase is a cell detachment solution that can be used to gently and effectively dissociate adherent cells from cell culture surfaces. It is a mixture of enzymes that can be used as an alternative to trypsin for subculturing cells.
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Lab products found in correlation
2 protocols using accutase medium
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Single-cell isolation from mouse embryos
2
Single-cell isolation and lysis protocol
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To remove the zona pellucida, the embryos were exposed to acidic Tyrode's solution (pH 2.5, Sigma, Cat#T1788) for 3-5 s and then washed thoroughly in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) (Sigma, Cat#A1933). Zona-free embryos were incubated for 10 min (for the 2-, 4-, and 8-cell stages) or 15 min (for the morula stage) in Accutase medium (GIBCO, Cat#A1110501), and then disaggregated by careful pipetting. Single blastomeres were placed into individual tubes containing 4 mL of lysis buffer (1.96 mL nuclease-free water, 1 mL of 10 mM dNTP mix (NEB, Cat#N0447), 0.1 mL of 40 U/mL RNase-inhibitor (NEB, Cat#M0314L), 0.04 mL 10% Triton X-100 (Sigma, Cat#T8787), and 1 mL of 10 mM modified oligo-dT primer (5 0 -AAGCAGTGGTATC AACGCAGAGTACT30VN-3 0 , where V is either A, C or G, and N is any base, Exiqon) or 0.5 mL of PBS for immediate preparation for RNA or DNA libraries, respectively.
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