title: GSM8163227: Sublibrary_THP-1_BRQ_CD11b_High; Homo sapiens; OTHER
library_construction_protocol: CRISPRi screens were performed using ER-Hoxa9, THP-1, or K562 dCas9-KRAB cells generated as above. For the genome-wide screens, libraries with 5 sgRNAs per gene (mCRISPRi-v2; the “top5” library from Addgene #1000000092) or 10 sgRNAs per gene (hCRISPRi-v2; both the “top5” and “supp5” libraries from Addgene #1000000090) were prepared as described in Palmer et al.119 with several modifications. Briefly, each plasmid library was electroporated into MegaX cells, maintaining at least 2000X representation, and plasmid was maxiprepped; the libraries were sequenced as described below to confirm even representation of guides. LentiX cells were transfected with plasmid as described above, and 48 hours later supernatant was collected and frozen in aliquots at -80ºC. ER-Hoxa9 and 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 (THP-1) or 150 million cells (ER-Hoxa9) were infected for 24 hours with lentiviral supernatant with 8 μg/ml polybrene, achieving an MOI of between 0.2 and 0.35. Lentiviral media was then replaced with fresh media, and 24 hours later, 2.5 μg/ml puromycin (ER-Hoxa9) or 1 μg/ml puromycin (THP-1) was added to select sgRNA-expressing 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 >90% for both replicates. At this point, puromycin was removed and 200 million (THP-1) or 50 million cells (ER-Hoxa9) were treated with DMSO or BRQ (500 nM for THP-1 cells). 48 hours later, cells were concentrated to 20 million cells/ml in fresh media and stained with antibodies and propidium iodide, and at least 15 million (THP-1) or 10 million (ER-Hoxa9) BFP-positive live cells from both the top and bottom 25% of flow marker expression (CD11b or CD14 for THP-1; CD11b or GFP for ER-Hoxa9) were sorted using a BD FACSAria, pelleted, and frozen at -80ºC for later processing. Aliquots of 15-20 million unsorted cells before and after treatment were also pelleted and frozen as controls for guide enrichment or dropout.  For the sublibrary screen, the top and bottom 300 hits of the THP-1 BRQ CD11b screen were selected, and all ten sgRNAs from the hCRISPRi-v2 library were incorporated into the sublibrary, along with ten sgRNAs for 894 randomly chosen genes and 87 hand-curated genes as well as 490 control sgRNAs, and oligos were synthesized as a pool (Twist Biosciences) (Table S4). Oligos were cloned into pU6-sgRNA EF1Alpha-puro-T2A-BFP (Addgene plasmid #60955) using methods described in Horlbeck et al. 2014120; briefly, oligos were amplified and digested with BstXI and BlpI, the 33bp insert containing the sgRNA sequence was size selected on a 10% PAGE gel and purified, and ligation into the linearized backbone was performed at a 1:1 molar ratio of insert to vector. Ligated plasmids were electroporated into MegaX cells at approximately 5000X library coverage, maxiprepped, and transfected into LentiX cells. 25 million THP-1 and K562 dCas9-KRAB cells were infected for 24 hours in duplicate with lentivirus and 8 µg/ml polybrene (MOI of between 0.25 and 0.35 for both cell lines), lentiviral media was replaced with fresh media, and 24 hours later puromycin selection was started at a concentration of 1 µg/ml. Cells were maintained at a concentration of between 250,000 and 1 million cells/ml and with a minimum of 500X library coverage throughout the selection, and puromycin was refreshed as above. After four days of selection, >90% of cells were BFP positive. 8 million K562 or THP-1 cells were treated with DMSO and 16 million THP-1 cells were treated with 500 nM BRQ for 48 hours (all screens performed in duplicate). 48 hours later, cells were concentrated to 20 million/ml and stained with antibodies and propidium iodide, and the top and bottom 25% of CD235a-expressing K562 cells or the top and bottom 25% of CD11b or CD14-expressing THP-1 cells were sorted and frozen at -80ºC for later processing. At least 2 million BFP+ live cells were collected per gate to achieve a minimum of 100X library coverage. Aliquots of 5 million unsorted cells before and after each treatment were also pelleted and frozen as controls for guide enrichment or dropout. To obtain amplicons for sequencing, genomic DNA from each cell pellet was extracted using the Machery Nagel Nucleospin Blood L kit (for genome-wide screens) or the Qiagen DNA Blood Mini kit (for sublibrary screens) following the manufacturer's instructions except for an overnight incubation at 70°C and elution of DNA into 200 μl 10mM Tris (pH 8.5). Guides were then PCR amplified using custom i7 and custom i5 primers indexed for each sample, with reactions split such that each 50 μl PCR reaction contained no more than 2.5 μg DNA in a 20 μl volume. For the THP-1 genome wide CRISPRi screens, each library was amplified using one of the p5 primers oBD370-375 and one of the p7 primers oBD367-368 (Table S5). For the ER-Hoxa9 genome-wide CRISPRi screens and all sublibrary screens, each library was amplified using one of the p5 primers oSV104-111 and one of the p7 primers oSV112-119 (Table S5). 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 1 μg of amplicon) was then purified using a double-sided selection with AMpure XP beads. 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. For the THP-1 genome-wide CRISPRi screen, we used an equimolar mix of custom read 1 primers, numbered oBD379-384 (Table S5). For the ER-Hoxa9 genome wide screens and all sublibrary screens, we used primer oBD582 (Table S5).