GSM8289874: After_CRISPRi_Rep_6; Homo sapiens; OTHER
For deep sequencing following screen, cells were collected from the top chamber by trypsinization. After harvesting and purifying genomic DNA, PCR was utilized to amplify sgRNA encoding regions. Then, using custom primers, sgRNA enconding regions were sequenced on Illumina HiSeq 4000 at high coverage, as previously described (Liu et al. Science 2017, Gilbert et al. Cell 2014). The U87-specific CRISPR inhibition (CRISPRi) library, derived from the human CRISPRi non-coding library (CRinCL) (Addgene #86542, a gift from Jonathan Weissman) by selecting sub-libraries that targeted all expressed lncRNAs in U87 (Liu et al. Science 2017), was used to generate the sgRNA pool used for screening. U87 cells expressing dCas9-KRAB-BFP were generated by lentiviral infection. pHR-SFFV-dCas9-BFP-KRAB was a gift from Stanley Qi & Jonathan Weissman (Addgene plasmid #46911; http://n2t.net/addgene:46911; RRID:Addgene_46911) (Gilbert et al. Cell 2013). Fluorescence associated cell sorting (FACS) was used 48 hours post-transduction to isolate cells stably expressing BFP (U87+dCas9-KRAB). The U87-specific CRISPR inhibition (CRISPRi) library, derived from the human CRISPRi non-coding library (CRinCL) (Addgene #86542, a gift from Jonathan Weissman) by selecting sub-libraries that targeted all expressed lncRNAs in U87, was used to generate the sgRNA pool used for screening (Liu Science 2017). TransIT-Lenti (Mirus, 6600) was used to package the plasmids for transfection in HEK293Ts and viral production was allowed to occur for 48 hours before harvesting. Filtered viral supernatant was then added to U87+dCas9-KRAB cells with polybrene (Sigma-Aldrich, TR-1003-G) at a final concentration of 4 µg/mL at an MOI < 0.3. Two days after infection, cells were treated with 0.75 µg/mL puromycin (Gibco, A1113803) for 3 days, allowed to recover for one day without puromycin, and used for assays the next day (T0), with cells being maintained at a coverage of 1000X. Two independent screen replicates were conducted, with cells collected at T0 and at the endpoint, and processed for sequencing on Illumina HiSeq 4000 and analyzed as previously described (Liu et al. Science 2017). A scatterplot of invasion phenotype of sgRNA of two independent replicates was generated, with the x and y-axes representing different screen replicates. Analysis output denoted p-value and effect sizes  for lncRNAs targeted in the screen. A specific screen score, incorporating these values, was calculated via x -log10 p value relative to negative internal control sgRNA targets as previously described(Liu et al. Science 2017, Gilbert et al. Cell 2014). These were plotted via volcano plot for selection of highest screen scores.