U2OSG3G7 (referred to as U2OS_DL1-ID mutant in the manuscript)
U2OS cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Life Technologies) and 1% penicillin/streptomycin (Invitrogen). Cells were harvested by trypsinization followed by two washes with ice-cold PBS. Genomic DNA from CRISPR/Cas9-targeted ZFP36L1 clones was extracted using Qiagen QiaAmp DNA mini kit (#51304) according to the manufacturers instructions and was used as a template for PCR amplification. Forward (5-CTGCTGGACAGAAAGGCAGT-3) and reverse (5-ATCCACAACGCTGAAGAGCGC-3) primers spanning the CRISPR-edited region were used for amplification of a 450bp fragment of Exon2 of zfp36l1. DNA library preparations, sequencing reactions, and initial bioinformatics analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). DNA library preparation, clustering, and sequencing reagents were used throughout the process using NEB Next Ultra DNA Library Prep kit following the manufacturers recommendations. Adapter-ligated DNA was indexed and enriched by limited cycle PCR. The DNA libraries were multiplexed and loaded on the Illumina MiSeq instrument according to manufacturers instructions.
GenlabU2OSG3G7