title: GSM3573383: Day28_Replicate1; Homo sapiens; OTHER
library_construction_protocol: Genomic DNA was isolated from cell pellets using a Genomic DNA Isolation Kit (Qiagen) sgRNAs were quantitated by amplification from genomic DNA using PrimeStar (Clontech) using primers flanking the sgRNA sequence for 20 cycles. The resulting product was column-purifed using a PCR purification kit (Macherey-Nagel). The entire product was amplified in a 7 cycle PCR to introduce Illumina sequencing primers and unique barcodes for eacht experiment. Libraries were quantified using the NEBNext Library Quantitation Kit (New England Biolabs) and mixed equimolar for sequencing. All designed library sgRNA sequences were prepended and appended with linker sequences (5' linker: CTTGTGGAAAGGACGAAACACC; 3' linker: GTTTAAGAGCTATGCTGGAAACAGC) to facilitate polymerase chain reaction amplification and to serve as invariant sequence overhangs for InFusion cloning. The oligonucleotides were synthesized (CustomArray) and delivered as a single oligonucleotide pool. The pool was amplified for 15 cycles (Forward primer: ATCTTGTGGAAAGGACGAAACA, Reverse primer: CTGTTTCCAGCATAGCTCTTAAAC) with CloneAmp HiFi (Clontech). The entire PCR reaction was resolved on a 2% agarose gel, and the product was retreived by gel slice isolation. A lentiviral vector pSICO-(F+E) was derived from the vector pSLQ1651 (Addgene plasmid # 51024) by insertion of a 1.9 kb BsmBI stuffer fragment between the U6 promoter and downstream tracrRNA. InFusion cloning was performed to assemble the sgRNA library into BsmBI-digested pSICO (F+E). Lentivirus was generated by transfection of lentiviral helper plasmids and the CRISPRi plasmid library into 293T cells, and lentiviral supernatant collected 48 hours afterwards. Supernatant was concentrated using Lenti-X Concentrator (Clontech) and frozen at -80°C in replicate aliquots.