Illumina MiSeq sequencing; Rapid High-Resolution Functional Mapping of Yeast Genomes by Saturated Transposition
Rapid High-Resolution Functional Mapping of Yeast Genomes by Saturated Transposition
ade2Δ strains were transformed with the pBK257 plasmid. These strains are phenotypically ade-, since the ADE2 gene borne on the plasmid is interrupted by the miniDs transposon. 1 liter of freshly prepared SC -Ura +2% Raffinose + 0.2% Dextrose is inoculated with ade2Δ cells freshly transformed with pBK257, directly scraped off the transformation plates, at a final OD600=0.15. The culture is grown to saturation for 18 to 24hr at 30°C. Cells are spun for 5min at 600x g, 20°C) and resuspended in their supernatant at a final OD600=37. 200 μl of this resuspension are plated on ~250-300 X 8.5cm plates containing 25 ml of SC +2%Galactose -Adenine using glass beads. Plates are incubated in closed but not sealed plastic bags for 3 weeks at 30°C. Clones in which transposon excision has led to the repair of the ADE2 gene on pBK257 start to appear after 10-12 days. The density of clones on the plate reaches 150-200 colonies/cm2, i.e.8000-11000 colonies/plates after 3 weeks. All colonies are then scraped off the plates using minimal volume of either water or SD +2% dextrose -Adenine, pooled, and used to inoculate 2L SD +2%Dextrose -Adenine culture at a density of 2.5 106 cells/ml, which is allowed to grow to saturation. This step is used to dilute any remaining ade- cells, which represent about 20% of the total number of cells, and ensures that each transposition event is well represented. For example, reseeding a 2.106 clones library in 2L at a density of 2.5 106 cells/ml will ensure that each clone is represented by ((2.500.000X1000X2)*0.8)/2.000.000= 2000 cells. The saturated culture is harvested by centrifugation (5 min, 2500x g, 4°C), washed with ddH2O, then cell pellets are frozen as ~500 mg aliquots.  A 500 mg cell pellet is resuspended with 500 μl Cell Breaking Buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 100 mM Tris-HCl pH8.0, 1 mM EDTA) and distributed in 280μl aliquots. 200 μl Phenol:Chloroform:Isoamylalcool 25:25:1 and 300 μl glass 0.4-0.6mm unwashed glass beads are added to each aliquot. Samples are vortexed for 10 min at 4°C using a Disruptor Genie from Scientific Industrial (US Patent 5,707,861). 200 μl TE are added to each lysate, which are then centrifuged for 5 min at 16100x g, 4°C. The upper layer (~400 μl) is transferred to a fresh tube, 2.5vol 100% EtOH are added and the sample mixed by inverted. DNA is pelleted for 5 min at 16100x g, 20°C. The supernatant is removed and the pellets resuspended in 200 μl RNAse A 250 μg/ml for 15 min at 55°C, 1000 rpm on a Thermomixer comfort (Eppendorf). 2.5 vol 100% EtOH and 0.1vol NaOAc 3 M pH5.2 are added and the samples mixed by inversion. DNA is pelleted by centrifugation for 5 min at 16100x g, 20°C. The pellets are washed with 70% EtOH under the same conditions, the supernatant removed completely, and the pellets let dry for 10 min at 37°C. The pellets are resuspended in a total volume of 100 μl water for 10 min at 55°C, 700 rpm on a Thermomixer comfort (Eppendorf).  DNA is run on a 0.6% 1X TBE agarose gel against a standard 1 kb GeneRuler, and quantified using FiJi. 500mg cell pellet should yield approximately 10 μg DNA. Sequencing involves the following steps: 1-Digestion of genomic DNA with two four-cutter restriction enzymes, 2-ligase-mediated circularization of the DNA, 3-PCR of the transposon-genome junctions using outward-facing primers. 4-Illumina-sequencing of the combined PCR products. 2x 2 μg of genomic DNA are digested in parallel in Non-Stick microfuge tubes (Ambion AM12450) with 50 units of DpnII (NEB #R0543L) and NlaIII (NEB #R0125L), in 50 μl for 16 hours at 37°C. The reactions are then heat inactivated at 65°C for 20 min and circularized in the same tube by ligation with 25 Weiss units T4 Ligase (Thermo Scientific #EL0011) for 6 hours at 22°C, in a volume of 400μl. DNA is precipitated overnight or longer at -20°C in 0.3 M NaOAc pH5.2, 1 ml 100% EtOH, using 5 μg linear acrylamide (Ambion AM9520) as a carrier, then centrifuged for 20 min at 16100x g, 4°C. Pellets are washed with 1 ml 70% EtOH, for 20 min at 16100 x g, 20°C. After complete removal of the supernatant, pellets are dried for 10 min at 37°C. Each circularized DNA preparation is then resuspended in  water and divided into 10X 100 μl PCR reactions. Each 100 μl PCR reaction contains: 10 μl 10X Taq Buffer (500 mM Tris-HCl pH9.2, 22.5 mM MgCl2, 160 mM NH4SO4, 20% DMSO, 1% Triton X-100 - stored at -20°C), 20 μM dNTPs, 1 μM primer #1, 1 μM primer #2 , 2.4 μl homemade Taq polymerase. PCR are performed in an MJ Research Peltier Thermal Cycler PTC-200 using the following conditions: Block: calculated - 95°C 1 min, 35X [95°C 30 sec, 55°C 30 sec, 72°C 3 min], 72°C 10 min. The 2X10 PCR reactions are pooled into one NlaIII-digested pool and one DpnII-digested pool. 100μl of each pool are purified using a PCR clean-up/gel extraction kit (Macherey-Nagel) according to the manufacturer protocol, with the following modifications: binding of DNA: 30s at 3000x g; elution: 30 μl of (10 mM Tris-HCl pH8.5, 0.1% Tween) are applied to the column and let sit for 3 min, then sample is spun for 1 min 11000x g at 20°C. The eluate is reapplied to the column and a second elution is performed under the same conditions. Purified PCR products are quantified by absorbance at 260 nm. On a 1% agarose gel, the product runs as a smear from 250bp to 1.2 kb, with highest density centered around 500bp. The 867 bp size band present in the NlaIII treated sample and the 465 bp size band present in the DpnII treated sample correspond to untransposed pBK257.
Experimental Factor: normal
growth condition