GSM7520496: HT29 Sox9-mKate2 Replicate 1; Homo sapiens; OTHER
Formed colonies were collected by trypsinization and resuspended in culture media. After regeneration culturing period (3-5 days), cell lines were tested by FACS analysis and site-specific PCR. The pCC_01 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE plasmid (Addgene #139086) was used for CRISPR screen and validation. Briefly the plasmid was digested with Esp3I (NEB#R0734) at 37oc overnight. The 13 kb band was purified with gel purification kit (Takara Nucleospin® Gel and PCR Clean-up Midi). Eighty-two base pair single stranded oligos were designed.31 base pair 5` constant sequence compatible with the linearized (Esp3I) pCC_01 vector (3` end of the U6 promoter) followed by the 19 bp sgRNA sequence adding an extra g base for the proper sgRNA transcription, followed by the 3`vector compatible (5` end of the sgRNA scaffold) sequence. Single stranded oligo pools were ordered from IDT (oPools™ Oligo Pools) and built into the digested PCC_01 vector using NEBuilder HiFi DNA Assembly Master Mix (E2621L, New England Biolabs) according to the manufacturer`s instruction. The list of all oligos can be found in the Supplementary Table 3. Assembled gRNA libraries were purified by AMPure XP magnetic beads (Beckman Coulter) Deep amplicon sequencing