GSM6448653: Genomewide_THP1_BRQ_CD11b_rep1_up; Homo sapiens; OTHER
The CRISPRi screen was performed as described (Palmer et al., 2018) with the following modifications, using THP-1 dCas9 cells generated as above and a genome-wide library with 10 sgRNAs per gene (Addgene #1000000090). The library was electroporated into MegaX cells (Invitrogen #C640003) and plasmid was maxiprepped (Qiagen) and sequenced as described below to ensure even representation of guides. THP-1 cells were always maintained between 250,000 and 1 million cells/ml, and two biological replicates were performed starting at the lentiviral infection step. 200 million cells were infected for 24 hours with lentivirus at an MOI between 0.25 and 0.35 with 8 μg/ml polybrene. Lentiviral media was then replaced with fresh media, and 24 hours later, 1 μg/ml puromycin was added to select library-containing cells. Media was exchanged and fresh puromycin was added 24 and 48 hours afterwards. Four days after puromycin treatment, viability was >90% and BFP positivity was >97% for both replicates, indicating that the selection was complete. At this point, puromycin was removed and 200 million cells were treated with 500 nM brequinar. 48 hours later, cells were concentrated to 20 million cells/ml in fresh media, and at least 15 million cells from both the top and bottom 25% of CD11b expression were sorted using a BD FACSAria, pelleted, and frozen at -80ºC for later processing. To obtain amplicons for sequencing, genomic DNA from each cell pellet was extracted using the Machery Nagel Nucleospin Blood L kit following the manufacturer's instructions except with an overnight incubation at 70C and elution of DNA into 200 μl 10mM Tris (pH 8.5). Guides were then PCR amplified using a universal i7 primer and custom i5 primers indexed for each sample, with reactions split such that each 50 μl PCR reaction contained no more than 2.5μg DNA in a 20 μl volume. Most pellets were split across either 16 or 32 PCR reactions depending on cell number. PCR was performed using the NEBNext Ultra Q5 2X Master Mix with the following cycling parameters: initial denaturation at 98°C for 2 minutes, then 24 cycles of denaturation at 98°C for 15 seconds and annealing/extension at 65°C for 75 seconds, then a final extension of 65°C for 5 minutes. All reactions for each pellet were pooled, and successful amplification was verified by running 10 μl of the PCR on an agarose gel to obtain an expected band between 250 and 260bp. 200 μl of the PCR reaction (roughly corresponding to 1μg of amplicon) was then purified using a double-sided selection with AMpure XP beads (Beckman Coulter). First, to remove genomic DNA, 0.5X volume of beads (100 μl) was added to the PCR reaction, mixed well, and incubated at room temperature for 10 minutes, and beads were placed on a magnet and supernatant was saved. Second, to remove primer dimers, 135 μl beads were added to 290 μl of supernatant (final bead volume of 1.2X), mixed well, and incubated at room temperature for 10 minutes, and beads were placed on a magnet and supernatant was discarded. DNA was then eluted from the beads using 20 μl of 0.1X TE (pH 8.5), quantified using Qubit and Bioanalyzer, and pooled for 1x75 sequencing on an Illumina NextSeq 500 using an equimolar mix of custom read 1 primers (p5_seq_primer, listed in Table S5).