Human crispr activation pooled library set a

Single guide RNA (sgRNA) sequences for non-targeting control and ACE2 were taken from the Weissman Human Genome-wide CRISPRa-v2 library (Addgene #83978). LRRC15 sgRNA sequences and additional ACE2 sgRNA sequences were taken from the Human CRISPR activation pooled library set A (Addgene #92379). Sense and antisense strands for each sequence were ordered as DNA oligonucleotides (IDT) with 5' overhangs of 5'-CACC-3' on the sense strand oligonucleotide and 5'-AAAC-3' on the antisense strand oligonucleotide. Oligonucleotides were annealed at 4°C for 16 h and pXPR-502 (Addgene #96923) was digested with Esp3I (ThermoFisher Scientific, ER0451) or BsmBI-v2 (New England Biolabs). sgRNA DNA oligonucleotide duplexes were ligated into the digested pXPR-502 backbone using T4 ligase (New England Biolabs) and incubated at 4°C overnight. NEB 10-beta competent E. coli (New England Biolabs) were transformed with 100 ng of each sgRNA construct by heat-shock, plated onto LBagar plates (Life Technologies) containing ampicillin (Sigma-Aldrich) and grown at 37°C. Individual colonies were picked, expanded in Luria broth (Life Technologies) supplemented with ampicillin and amplified constructs were harvested using either ISOLATE II Plasmid Mini Kit (Bioline) or PureYield Plasmid Maxiprep System (Promega Corporation).

MegaX DH10B T1 R Electrocomp™ Cells (ThermoFisher Scientific) were electroporated with 400 ng Human CRISPR activation pooled library set A (Addgene #92379) and left to recover in (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted November 15, 2022. ; https://doi.org/10.1101/2021.11.09.467981 doi: bioRxiv preprint Recovery Medium for 1 hour at 37°C. Cells were then spread on 600 cm 2 LB-agar plates supplemented with carbenicillin (Merck) and incubated at 37°C for 16 hours. All colonies were scraped, collected and processed using the PureYield Plasmid Maxiprep System (Promega Corporation). The concentration of the plasmid library was determined via Nanodrop (ThermoFisher Scientific).