title: NextSeq 500 sequencing; Systematic identification of G-quadruplex sensitive lethality by genome-wide genetic screening
design: Systematic identification of G-quadruplex sensitive lethality by genome-wide genetic screening
library_construction_protocol: Cell Lines: HT1080, A375 (ATCC) and Plat-A (Cell Biolabs Inc) cell lines were cultured in DMEM medium (Gibco, cat #41966029) supplemented with 10% (v/v) heat inactivated FBS (Gibco, cat #10500064). All cell lines were grown at 37 °C in a 5% CO2 humidified atmosphere and regularly checked to be mycoplasma-free by RNA-capture ELISA. Cell line genotypes were certified by STR profiling. Quantification of live cell numbers: Throughout, live cell numbers (e.g. for plating cells for the cell titre glo assays, the screens and Incucyte experiments) were determined using the Muse Cell Analyser, 'Count & Viability' assay according to manufacturer's instructions. Cells were diluted either 1:10 or 1:20 in 'Muse Count & Viability kit' solution, to give a viable cell concentration of 1-2 x 106 cells/mL, with 'Events to Acquire' parameter set at 1000 events. Determination of G-quadruplex ligand concentration for shRNA screens: Throughout, PDS and PhenDC3 were used as 50 mM stocks, dissolved in DMSO. GI20 values were calculated by treating A375 and HT1080 cells with serial dilutions of PDS and PhenDC3 for 96 h and determining cell death via a Cell Titre Glo assay (Promega) according to manufacturer's protocol. Each serial dilution was replicated 4 times for 2 cell-seeding densities (1000/1500 cells per well). For both densities, curves were plotted averaging the 4 replicates in GraphPad Prism using a Non-Linear regression model, “dose-response – inhibition” equation [log(inhibitor) vs. normalised response – variable slope] and GI20 values calculated. The GI20 concentrations used represent an average for three separate assays per cell line and yielded the following concentrations used for the screens - A375: 10 μM PhenDC3 and 1.5 μM PDS; HT1080: 1 μM PhenDC3 and 0.5 μM PDS. Composition of and recombinant DNA production from shRNA libraries: The genome-wide screen uses the Transomic LMN shRNA library against the human protein coding genome, consisting of 113,002 total shRNAs, split between 12 pools for ease-of-handling (approximately 10,000 shRNAs per pool) with an average number of 6 optimised hairpins per gene. The pilot screen uses one pool (8) of the Transomic LMN shRNA library used in the genome-wide screen. The G-quadruplex focussed screen consists of a custom shRNA pool (Transomic) with the same LMN vector (8,018 shRNAs); this includes 1247 genes (7,436 shRNAs) uncovered in the GW screen (751 sensitisers and 496 upregulated genes), 116 other 'interesting' genes from the literature (439 shRNAs) and shRNAs targeting 37 olfactory receptors as non-targeting controls (143 shRNAs). In this custom pool, unlike the commercially available genome-wide library, we capped the number of shRNAs at 7 per gene (SF1B). The backbone of both libraries contains NeoR and ZsGreen markers to allow monitoring of infected cell lines by Geneticin (Gibco) selection and fluorescence (MacsQuant) respectively. Both libraries were provided as glycerol stocks. The bacterial density of these was determined by calculating the colony forming units (CFU) of dilutions of the original stock after plating on agar (O/N, 37 0C, 100 μg/ml ampicillin). Then, glycerol stocks were thawed completely and sufficient volume was taken (based on CFU) to ensure a minimum of 1000-fold hairpin representation, and inoculated in gigaprep bacterial cultures (LB media + 100 μg/ml ampicillin). Plasmid DNA was isolated from these cultures using ZR Gigaprep kit D4057 (Zymo research) according to manufacturer's protocol and DNA was quantified by Nanodrop Onec.  shRNA stable cell line creation: For the genome-wide screen, each pool was treated independently, necessitating the creation of 12 different polymorphic cell lines each containing an average of 10,000 shRNAs, for both HT1080 and A375, per replica (3 replicas, 36 polymorphic cell lines). Virus was produced using the Platinum-A packaging cell line and calcium phosphate transfection. 24 h after plating Platimum-A cells, media was replaced with DMEM medium supplemented with 1 % PenStrep and 10% (v/v) heat inactivated FBS. Then plasmid DNA isolated from the bacterial gigapreps were co-transfected with pCMV-VSV-G plasmid according to manufacturer's protocol. Cells were also transfected with Pasha/DGCR8 siRNA (Qiagen) to increase viral titre. Immediately before adding the DNA-Pasha-transfection mixture, chloroquine (lysosomal inhibitor) is added to the plates at a final concentration 2.5 uM. 48 h after transfection virus was harvested and filter sterilised (0.45-μM) and stored at 4 0C for a maximum of 7 d. Viral titre was determined by performing mock infections with the experimental cell line and quantifying fluorescent cells, via flow cytometry (MacsQuant) 48 h after infection. For both the genome-wide and focussed-screen, 3.6 x 106 target cells were infected at a viral volume predicted to cause 30 % infection (MOI 0.3) to minimise multiple shRNA integrations per cell. This provides approximately 10 x 106 shRNA expressing cells (1000-fold shRNA representation). The virus was diluted in serum free media plus polybrene (8 μg/mL). Infections were carried out in triplicate, and treated as independent replicates hereafter. 48 h after infection cells were selected in 800 μg/ml (HT1080) and 1000 μg/ml (A375) geneticin for 7-9 days, concentrations determined via 7 day geneticin toxicity curves. Cell culture process for genome-wide and focussed pool of shRNAs: Following complete selection, a reference timepoint was harvested (t0) and cells were split into 3 x 15 cm plates per replica: PDS, PhenDC3 and DMSO vehicle control, each containing 8-10 x 106 cells to maintain 1000-fold hairpin representation. Every 72 h, cells were trypsinised, counted to determine the number of population doublings, and 10 x 106 (A375 GW and focussed) or 8 x 106 (HT1080) cells per replica replated in fresh drug/DMSO and media. The volume of DMSO used in the 'vehicle' condition is equal to the volume for 10 uM PhenDC3. The remaining drug treatments were also supplemented with DMSO to match this volume and keep the DMSO concentration equal between treatment, cell lines and screens. Therefore at all times, sufficient cell numbers were used so that a minimum of 1000 or 800 cells per shRNA were maintained (A375 and HT1080 respectively). A final timepoint (t15) was harvested after 15 population doublings. For each pool of the genome-wide screen, 12 samples were generated (t0, DMSO t15, PDS t15, PhenDC3 t15; 3 replicas each). Therefore, 144 samples of 10 x 106 cells were generated to cover the entire screen. For the focussed screen, for each cell line, 12 samples were generated (t0, DMSO t15, PDS t15, PhenDC3 t15; 3 replicas each). All PCR and Sequencing oligos were ordered from Sigma. Cell pellets (10 x 106 cells) were resuspended in PBS and gDNA extracted using QIAmp DNA Blood Maxi Kit (Qiagen) according to the manufacturer's spin protocol, eluted in a final volume of 1200 μl and quantified by Qubit DNA HS Assay Kit. shRNA inserts were PCR-amplified from all gDNA in each sample, in multiple 50 μl reactions each using 1.5 μg gDNA, using KOD Hot Start DNA Polymerase and the following reagents (Merck Millipore): 5 μl 10 x buffer, 5 μl 2 mM each dNTPs, 4 μl MgSO4 (25 mM), 1.5 μl polymerase, 4 μl DMSO. Forward and reverse primers flanking the loop and antisense sequence of the hairpin region (Sigma) were used at a final concentration of 300 nM. PCR was performed under the following conditions: 98 oC for 5 min, then for 25 cycles of 98 oC for 35 s, 58 oC 35 s, 72 oC for 35 s, followed by 72 oC for 5 min. 1.2 mL of pooled PCR reaction were cleaned-up using QIAquick PCR purification kit (Qiagen) according to manufacturer's protocol. 2 μg of this purified PCR product were PCR amplified in a second PCR step, using forward and reverse primers flanking the antisense sequence of the hairpin and containing the P5 and P7 flowcell adapters respectively. The PCR was performed in 8 x 50 μL reactions each containing 500 ng template DNA. The reverse primer contains TruSeq adapter small RNA Indexes to allow multiplexing of the samples, a 6 nucleotide barcode, denoted 'nnnnnn' below. PCR reagents were used as for the first PCR, with the exception of the primers, which were used at a final concentration 1.5 μM. The second PCR was performed under the following conditions: 98 oC for 5 min, then for 25 cycles of 94 oC for 35 s, 52 oC 35 s, 72 oC for 35 s, followed by 72 oC for 5 min. 200 μl of pooled secondary PCR product was cleaned up as previously and the desired product (~340 bp) was extracted using BluePippin 2 % Internal Standard Marker Kit (DF marker 100-600 bp; Sage Science), according to Manufacturer's protocol using a broad range elution (300-400 bp). Individual samples were quantified using KAPA library quantification kit, Illumina platforms, BioRad CFX96 Instrument, No Rox according to manufacturer's protocols and diluted to 4 nM in RNAse free water. For the GW screen samples, 24 libraries (2 pools) and for the focussed screen samples, 30 libraries (both cell lines) were combined to create a pooled 4 nM stock, with each sample having a unique TruSeq adapter.
attributes.tag: Experimental Factor: cell line
attributes.value: A375
attributes.tag: Experimental Factor: RNA interference
attributes.value: Transomic LMN shRNA library against the human protein coding genome - Pool 12
attributes.tag: Experimental Factor: compound
attributes.value: DMSO
attributes.tag: Experimental Factor: dose
attributes.value: volume of DMSO equal to the volume for 10 M PhenDC3
attributes.tag: Experimental Factor: time
attributes.value: 15 cell population doublings