Synth a freeze

CryoStor CS10 and CS5 (BioLife Solutions), and Synth-a-Freeze (SAF; Thermo Fisher) were used in accordance with manufacturer’s instructions or as previously described (Newman and Kaur, 2015 ).
To freeze, dissociated cells from a fresh dissection were pelleted, resuspended in ice-cold freezing media at a density of 6 × 10⁶ cells/ml, and aliquoted into cryovials on ice. Each aliquot contained 1.5 × 10⁶ cells. Cryovials were placed in an isopropanol cell freezing container pre-chilled to 4°C, stored at -80°C for 2 d, and then transferred to the vapor phase of liquid nitrogen for long-term storage.
To thaw, to reduce shear stress during handling, cells were transferred and resuspended using a P1000 tip cut to widen the diameter to ∼2 mm. Cryovials were rapidly thawed in a 37°C water bath until a small ice crystal remained. Cells were then gently resuspended by drop-wise addition of warm Plating Media to the cryovial with periodic swirling. This volume was gently transferred to a conical tube containing 10× volume warm Plating Media. The cell suspension was pelleted by centrifugation at 150 × g for 5 min and resuspended in 1 ml fresh Plating Media by pipetting ∼15 times with a cut P1000 to break up cell clumps. Cells were counted using Trypan blue and a hemocytometer and plated as required.

For gene targeting WTC cells at passage 22 were dissociated in 1 mL Accutase, collected into a 15-mL Falcon, centrifuged (160 × g, 3 min, RT) and counted manually. Three million cells were resuspended in 100 μL of Lonza Amaxa Primary P3 nucleofection solution with 3 μg vector DNA for mMaple transgene insertion and 1 μg of a plasmid expressing the guide RNA and CRISPR-Cas9 (pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang, Addgene plasmid # 62988) and subjected to nucleofection following the manufacturer's instructions for the Lonza Amaxa Primary P3 Kit (V4XP-3024, Lonza). After nucleofection cells were replated onto a 6 well plate in mTser Plus supplemented with 10 μM ROCKi Y-27632 (Lonza-PeproTech, 1293823-B) for 24 h. After 72 h cells were incubated with mTeSR Plus containing 0.5 ug/ml of Puromycin (Life Technologies, A1113803) for 72 h. After further 5 days of culture cells were detached with Accutase and single cell cloned. After 2 weeks of expansion individual clones were screened and frozen in Synth-a-Freeze (Thermofisher, A1254201).