Genome-wide CRISPR Knock-out screen of LIMD1-/- isogenic and parental A549 cell line
Genome-wide CRISPR Knock-out screen of LIMD1-/- isogenic and parental A549 cell line
Cells were split twice per week at the indicated timepoints. Every time a cell number corresponding to roughly 500-fold coverage of the library was pelleted, washed once with D-PBS and then stored at -20°C before processing.  Genomic DNA was extracted using the Qiagen QIAamp DNA Blood Maxi kit (order number 51194) according to the manufacturer's protocol.   https://www.qiagen.com/de-us/products/discovery-and-translational-research/dna-rna-purification/dna-purification/genomic-dna/qiaamp-dna-blood-kits?catno=51194  NGS amplicon libraries were generated with a two-step PCR protocol. First, the lentiviral cassette containing the gRNA, was amplified from genomic DNA with specific primers that also contain Illumina specific adapter sequences. In the second PCR, 8 nucleotide Nextera indices were added using Indexing primers with complementary sequences to the 1st PCR primers and contained P5 and P7 Illumina compatible sequences necessary for cluster generation. Final amplicon NGS libraries were cleaned up using AMPure XP beads. 