DOI: 10.7924/r4df7089v
title: Data from: The essential clathrin adaptor protein complex-2 is tumor suppressive specifically in vivo

description: in vivo and in vitro CRISPR/Cas9 screen
Library design
The sgRNA library was designed to target all phosphatidylinositol synthetases, kinases, phosphatase, lipases and regulatory genes with 5 sgRNA each based on a previous report72, in addition 100 control (ctrl) sgRNAs totaling 660 sgRNAs.  See Supplemental Data 1 for sgRNA library details.

Library cloning
Pooled oligos encoding sgRNA with 5' and 3' adaptor sequences (CustomArray Inc) were diluted 1:10 in water and amplified using Phusion Hotstart Flex master mix (New England Biolabs Inc) with the ArrayF and ArrayR primers (IDT).
ArrayF- -  TAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG
ArrayR- -  ACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC
sgRNA amplicons were cloned into lentiCRISPR v2-Blast, a gift from Mohan Babu (Addgene plasmid # 83480; http://n2t.net/addgene:83480; RRID:Addgene_83480).  5 ¬µg of the lentiCRISPR v2-Blast vector was digested with FastDigest Esp3I (Thermo Fisher Scientific) enzyme and electrophoresed on a 1% agarose gel.  The ~11kb was excised from the gel and purified using a QIAquick Gel Extraction Kit according to the manufacturer's instructions (Qiagen).  The amplified library was assembled into the restriction-digested vector using Gibson Assembly Master Mix (New England Biolabs Inc) and electroporated into library competent E. coli (Lucigen Corp).  Library plasmid DNA was purified using a HiSpeed Plasmid Midi Kit according to the manufacturer's instructions (Qiagen) and library coverage was validated by Illumina NextSeq 500 single-end sequencing. 

Lentivirus production
HEK293T cells were cultured to 70-80 % confluency and transfected with lentiviral packaging system at the ratio of 1:1:2 for psPAX2:pVSVg:lentiCRISPR v2 Blast using Fugene6 (Promega) transfection reagent.  After 48 to 72 hours, media was harvested from cells, centrifuged at 400 RCF for 3 minutes and filtered using a 45 ¬µm pore filter (VWR) to remove cell debris.  Virus was either used immediately or snap frozen for future use. 

Cell infection
For each replicate, 1.2 x 107 cells were infected at an MOI of 0.3 to ensure that most cells were infected by a single lentiviral particle.  Each infection was performed across 6 wells of a 6-well plate in 1 ml of the appropriate media containing virus at a predetermined concentration to reach the targeted MOI in the presence of 8 ¬µg/ml polybrene (MilliporeSigma) and spun at 800 x g for 1 hour.  After 6 hours of culture at 37 ¬∞C, cells were washed in 1x Phosphate Buffered Saline (PBS; Sodium Chloride, 0.137 M; Potassium Chloride, 0.0027 M; Sodium Phosphate Dibasic, 0.01 M; Potassium Phosphate Monobasic, 0.0018 M), and the media was replaced.  18 hours later, each infection was pooled into a 15 cm culture dish and selected with 5 ¬µg/ml blasticidin (Thermo Fisher Scientific), except for PANC-1 cells, which were selected with 20 ¬µg/ml blasticidin.  Cells were continuously passaged and selected for 1 week after initial infection at which point 1 x 106 cells were pelleted and snap frozen as the initial sample.  1 x 106 cells were continuously passaged for 2 weeks for in vitro samples.  5 x 106 cells were transplanted into 1 male and 1 female Fox Chase SCID/Beige mouse (details below) (Charles River).   In vitro samples were pelleted and snap frozen after 2 weeks of growth. 

Xenograft transplant
For each transplant, 5 x 106 cells were resuspended in 200 ¬µl sterile PBS and kept on ice until injection (less than 2 hours).  Mice were anesthetized in a bell jar with diffuse isoflurane, their injection site sterilized with iodine, and subcutaneously injected with 200 ¬µl cell suspension in the right flank using a 30 G needle (BD).  Mice were monitored hourly for 24 hours, then afterward, three times a week for tumor growth and morbidity and moribundity.  Mice were humanely euthanized by CO2 asphyxiation followed by decapitation upon a tumor size of ~1 cm3 or upon signs of moribundity, and tumors removed and snap frozen during necropsy.

gDNA isolation, sgRNA amplification, and sequencing
Cell samples were lysed in 1 ml DNAzol by vigorous pipetting according to the manufacturer's protocol (Molecular Research Center inc.).  DNA was precipitated in 0.5 ml 200 proof ethanol (VWR).  30 to 50 mg of tumor samples were lysed in 5 ml DNAzol by a motorized tissue homogenizer (Kinematica AG PT1200), centrifuged at 3,200 g for 15 minutes at 4 ¬∞C, and transferred to a new tube.  gDNA was then precipitated by 2.5 ml 200 proof ethanol.  Precipitated DNA was pelleted by centrifugation at 20,000 g, washed twice in 75% ethanol.  gDNA was solubilized in 0.5 ml or 1.0 ml of water for cell and tumor samples, respectively.  To reduce PCR bias, amplification of sgRNA sequences from each sample gDNA was performed in triplicate and pooled at each step.  First round of PCR amplification was performed using 50 to 100 ¬µg gDNA, Hotstart Taq (Takara), and 1 ¬µM CRISPR F1 and CRISPR R1 primers (IDT).
CRISPR F1 - AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG
CRISPR R1 - TCTACTATTCTTTCCCCTGCACTGTtgtgggcgatgtgcgctctg
Thermal cycles consisted of 95 ¬∞C for 5 minutes, followed by 18 cycles of 95 ¬∞C for 30 seconds, 60 ¬∞C for 30 seconds, 72 ¬∞C for 30 seconds, and a final cycle of 72 ¬∞C for 5 minutes.  Amplicons from the same samples were pooled and a second round of PCR amplification was performed to add sample barcode and Illumina adaptor sequences using 20 ¬µl of PCR1 and 0.5 ¬µM  CRISPR R2 and barcoded forward primers. See Supplemental Data 2 for barcode information. 
CRISPR R2 - CAAGCAGAAGACGGCATACGAGATGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTtctactattctttcccctgcactgt
Thermal cycles consisted of 95 ¬∞C for 5 minutes, followed by 20 cycles of 95 ¬∞C for 15 seconds, 58 ¬∞C for 30 seconds, 72 ¬∞C for 30 seconds, and a final cycle of 72 ¬∞C for 5 minutes.  Amplicons from the second round of PCR were pooled at equal concentrations, were purified using Ampure XP Beads according to the manufacturer's protocol (Beckman Coulter).  Purified amplicons were subjected to Illumina NexSeq 500 75bp single end sequencing.  

CRISPR screen sequencing analysis
Sequencing reads were de-multiplexed using fastx_barcode_splitter (v0.0.13) to identify inline barcodes and aligned to the reference library using bowtie (v1.0.0), allowing for 0 mismatches.  Sample sgRNA read counts were first normalized to total reads/sample.  sgRNA fold-change values were then calculated by comparing in vitro and in vivo sample sgRNA counts to their paired initial sample sgRNA count.  Gene target enrichment scores were then calculated by averaging the 5 sgRNA fold-change values within a sample.  Average sample enrichment fold-changes distributions were normalized by first zeroing each sample on the average enrichment of non-targeting control sgRNAs and then calculating a z-score.  To compare in vitro enrichment to in vivo enrichment, replicate sample z-scores were averaged and compared by T-test.  Resultant p-values were adjusted by calculating false discovery rates.