INCERTIDUMBRE POLÍTICA

Gene-specific RNAi inserts were designed using a web-based tool, RNAit (Redmond, Vadivelu and Field, 2003), and ranged between 450 and 650 base pairs. Such restricted size, in addition to the Gateway® flanks, AttL1 and AttL2, were exploited to design a universal PCR, which enabled homogenous

enrichment of the RNAi targets from genomic DNA and increased efficiency of the sequencing reaction (Figure 3-2C). It was critical to use a high fidelity polymerase to ensure amplified sequences were indistinguishable from their templates, so we could unequivocally assign sequencing reads to the original templates.

After alignment in CLC genomics workbench, AttL1 and AttL2 displayed in nearly identical sequences, differing in only 4 bases. Based on that, a variety of primers were designed, trying to achieve optimal PCR amplification (Figure 3-4A): two forward primers (Fw1 and Fw2) binding to AttL1; two reverse (Rv1 and Rv2) hybridizing in the stuffer region; and two reverse (Rv3a and Rv3b) binding to AttL2, Figure 3-4B.

Figure 3-4. PCR optimization for enrichment in the RNAi insert.

A. (Above)Sequence alignment of the AttL sites within the universal Gateway®

flanks. Highlighted on top, forward (Fw) and reverse (Rv) primers designed exploiting the single nucleotide conflicts between sequences. (Below) Schematic representation of the RNAi cassette (see Figure 3-1) indicating binding sites for all the oligonucleotides tested. B. List of all primers tested during the

optimization process. C. Phusion® high fidelity polymerase reaction (60 oC annealing/30 cycles) with different combinations of primers. Depending on the template, lanes are labelled: G=gDNA from an RNAi line (sTL0082); P= DNA from its untransfected plasmid (pTL50); B=genomic DNA from parental 2T1s; W=water control. D. Fw1/Rv2 PCR with Phusion® high fidelity polymerase (60 oC

annealing/30 cycles). Lanes labelled as in (C) with G*= positive control for PCR with gDNA from sTL0082 using primers to amplify the kinase domain of

Tb927.9.10920. E. Fw1/Rv4 gradient PCR using pTL50 DNA and Phusion

polymerase for 30 cycles, annealing temperature highlighted above each lane. F. Q5 high fidelity polymerase reaction (61o_{C annealing/30 cycles) using Fw1/Rv4}

and single primer Fw1. Lanes labelled as in (B) with 5xG= gDNA from 5 RNAi lines selected from the library. G. Fw1 (OL4161) Q5 high fidelity polymerase reaction (61 oC annealing/input kinase pool as a template) increasing number of PCR cycles.

All combinations were tested with Phusion High Fidelity polymerase at 60 oC. As a DNA template we used a plasmid from the library (pTL50) and genomic DNA (gDNA) from the cell line generated upon pTL50 transfection into 2T1 cells (sTL0082), Figure 3-4C. The construct and cell line targeted ATR by RNAi, a

protein kinase involved in DNA repair, which has been validated previously somewhere else: the plasmid through restriction digestion and sequencing; and the cell line via antibiotic resistance, RNA/protein depletion, and decrease in resistance to genotoxic effects caused by methyl methanesulfonate (Stortz, PhD thesis, 2016). As a control for PCR, primers were also tested against gDNA from parental 2T1 cells, and water. We only could obtain a single defined PCR product that matched the predicted 628-bp with Fw1/Rv2. This size includes a small AttL1 portion (starting from Fw1), plus the RNAi target with the complete Attl2 flank and the section of the stuffer before Rv2 binding site. PCR amplification was only possible with the plasmid template, not with gDNA.

In order to verify that gDNA from sTL0082 was not degraded we used PCR

conditions and primers validated to amplify the kinase domain of Tb927.9.10920 (kindly provided by Nathaniel Jones) in parallel to the Fw1/Rv2 PCR, Figure 3-4D. This time, more DNA was loaded in the gel in order to increase resolution. We observed, on the one hand, that genomic DNA was fine because a band could be identified at the expected size for the genomic control (882 bp), and on the other hand, that a secondary amplicon was produced with Fw1/Rv2. After sequencing the secondary band, we found it corresponded to the 488 bp-long product of Fw1 (OL4161) acting both as forward and reverse primer, binding to AttL2 with a single mismatch at the 3´end (G instead of A), Figure 3-4B.

To solve this problem, a reverse primer targeting the site where Fw1 hybridized unspecifically to AttL2 was designed (Rv4). This enabled production of a single band, and 61 oC was selected as the annealing temperature after a gradient PCR, using pTL50 as a template, Figure 3-4E.

Applying the same conditions with Fw1/Rv4 and the more efficient Q5 High fidelity polymerase, we were finally able to PCR amplify the RNAi target from sTL0082 gDNA alone and pooled with gDNA of other 5 lines included in MSTL1, which targeted: Tb927.2.2260 (584 bp PCR product), Tb927.8.7450 (490 bp), Tb927.11.850 (428 bp), and Tb927.1.1000 (437 bp). In parallel, a PCR was

performed in the same conditions with Fw1 alone, concluding that its efficiency was better than using Fw1/Rv4 combined, Figure 3-4F.

In Figure 3-2C, which illustrates the overall approach, we displayed results of PCR amplification with Fw1 of four different samples: in vivo uninduced mouse (1), in vivo induced mouse (2), gDNA from sTL0082 (3), and input library pool (4).

It was necessary to adjust the optimal number of cycles for the single primer PCR in order to avoid saturation levels. This can be caused by accumulation of double stranded DNA produced during the exponential phase, which saturates the polymerase. Against common thinking, saturation does not happen due to an exhaustion of reagents (e.g. primers, dNTPs) (Kainz, 2000). Although primers were designed to bind in the universal flanks, slight differences in size and GC content may cause some amplicons to be favoured over others. Saturation would overenrich the most physio-chemically privileged RNAi targets in detriment of the less favoured, potentially compromising the basis of the RITseq screen, even when this compares induced/uninduced reads per gene ID. 28 cycles was chosen as this produced enough enrichment while far from saturation (Figure 3-4G) Addition of 6-nucleotide tags to the single primer used to perform the PCR (Fw1= OL4161) permitted expanding our multiplexing capacity and reducing overall costs of the experiment. Pooling equal masses of PCR products generated with distinct barcoded primers, from gDNAs purified from different experimental conditions or replicates, permitted creating a single sequencing sample. Reads could be assigned later to the original experimental condition in silico (Figure 3-5).

Figure 3-5. PCR bar-coded strategy for sample enrichment of the RNAi target. A. Schematic representation of the strategy. 6-mer labels added to OL4161

permits pooling equal masses of PCR products obtained from different experimental conditions (cond.1, 2, 3) and use of a single set of Illumina sequencing adapters. B. 1% agarose gel showing homogeneous amplification of gDNA purified from the input library with the 14 selected barcoded versions of OL4161. Sequences for each of the primers is depicted in the table.

The PCR enrichment strategy required 13 plasmids and their respective cell lines to be remade so that they could include the universal Gateway® sites flanking the RNAi cassette. The original plasmids had been made using traditional restriction digestion-ligation cloning and would not have been suitable for PCR amplification of the RNAi target sequence by OL4161. As with the rest of the library (Jones et al., 2014), alamar blue phenotypes after 72 h of RNAi induction were also analysed (Table 3-2). Among these lines a few showed a loss of fitness: the 2 clones made for CRK3, KKT10 and AGC1, one clone for KKT19, PDK1 and the orphan kinase Tb927.11.6690.

Table 3-2. Alamar blue (AB) ratios (Tet+/Tet-) after 72h for cell lines remade in the Gateway® system to enable PCR enrichment in the pooled library.

Plasmid Gene ID PK name Cell line Mean AB

(Tet+/Tet-) SD pTL21 Tb927.10.4990 CRK3 sTL0024 0,511 0,011 sTL0097 0,516 0,023 pTL23 Tb927.6.2250 AGC (RAC) sTL0031 0,997 0,022 sTL0034 0,974 0,203 pTL236 Tb927.10.16160 Orphan sTL590 1,049 0,040 sTL591 0,996 0,036 pTL237 Tb927.3.3290 Orphan sTL593 0,956 0,095 sTL594* 1,031 0,031 PTL234 TB927.11.12420 CMGC/CLK2 (KKT19) sTL538 1,023 0,058 sTL596 0,820 0,122 pTL128 Tb927.10.14300 STE (STE11) sTL616 0,984 0,045 sTL617 0,976 0,022 PTL233 TB927.11.12410 CMGC/CLK (KKT10) sTL535 0,618 0,009 sTL594 0,721 0,172 PTL244 Tb927.3.2440 AGC1 sTL618 0,606 0,012 sTL619 0,801 0,019 pTL235 Tb927.7.3580 NEK-11 sTL708 0,945 0,002 sTL709 1,020 0,042 pTL238 Tb927.3.3080 NEK-6 sTL710 1,042 0,028 sTL711 0,985 0,014 pTL240 Tb927.8.1670 NEK-13 sTL712 0,979 0,016 sTL713 0,994 0,036 pTL241 Tb927.9.4910 AGC (TbPDK1) sTL672 0,896 0,191 sTL714 1,004 0,044 pTL243 Tb927.11.6690 Orphan sTL715 0,502 0,014 sTL716 1,047 0,043

SD= standard deviation. Mean AB highlighted in red= loss of fitness phenotypes. PK = Protein kinase.