Generation of stable cell lines expressing 3xFLAG-CDK12

Dear Chulai,

Below is our published method. It is hard to troubleshoot your experiment with limited information you provided. Please contact me by email if you need more information from us.

Ting Han

hanting@nibs.ac.cn

An sgRNA sequence (5’-GCCCAATTCAGAGAGACATG-3’) targeting the genomic region immediately downstream the CDK12 start codon was cloned into the PX458 vector (Addgene 48138). The 3xFLAG knock-in repair template was constructed in a pTOPO-TA vector (Mei5bio, Beijing, China) containg a BSD-P2A-3xFLAG sequence flanked by two 500 bp homology arms matching upstream and downstream sequences of the CDK12 genomic locus. For endogenous tagging of CDK12, 1 million A549 cells were nucleofected (using 4D-Nucleofector, Lonza, Basel, Switzerland) with 1 µg of PX458-sgCDK12 and 1 μg of the repair template. Selection with 30 μg/ml of blastistin was performed until clones appeared. Multiple clones were isolated and successful integration of N-terminal 3xFLAG tag was validated by western blotting with anti-FLAG-HRP (Sigma-Aldrich, MO, USA, A8592, 1:10,000).