2.5. E. coli ORFeome capture
The LASSO libraries were hybridized on E. coli genomic DNA. The hybridization was performed in 15 μl of 1× Ampligase DNA Ligase Buffer (Epicentre) containing 250 ng of unshared E. coli K12 total genomic DNA and 5 ng LASSO probe library. In the hybridization volume, the concentration of E. coli chromosomes was approximately 10 pM. The concentration of individual LASSO probes was approximately 14 pM (44 nM for the complete LASSO). The solution (15 μl) containing the MIP or LASSO probe pool and the E. coli DNA was denatured for 5 min at 95 °C in a PCR thermocycler (Eppendorf Mastercycler), then incubated at 65 °C for 60 min. After hybridization, 5 μl of the freshly prepared gap filling mix was added into the hybridization solution while maintaining the reaction at 65 °C in the thermal-cycler. Gap filling and ligation was performed for 30 min at 65 °C. After capture, the DNA samples were denatured for 3 min at 95 °C, and the temperature was reduced to 37 °C. Next, 4 μl of linear DNA digestion solution was added immediately. Digestion was performed for 1 h at 37 °C, followed by 20 min at 80 °C.
The gap filling mix composition for 1ml volume of gap filling mix was as follows: 791 μl of PCR grade water, 100 μl of glycerol, 4 μl of 10 mM dNTPs, 1 μl of Ampligase DNA Ligase (100 U μ l−1), 4 μl of Omni Klentaq LA, 100 μl of 10× Ampligase DNA Ligase Buffer.. This gap filling mix could be stored at -20°C for up to three months.
The linear DNA digestion solution (volume: 48 μ l) was composed of 24 μl of nuclease-free water, 6 μl of Exonuclease I (20 units μ l−1), 6 μ l of Exonuclease III (100 units μ l−1), 6 μl of Lambda Exonuclease (5 units μl−1) and 6 μ l of Exonuclease VII (10 units μl−1) (all from NEB).
2.6. Capture of human ORFs using single LASSO probes from human cDNA .
To capture of human ORFs, was performed using 50 ng Human Reference cDNA (Zyagen) and 0.02 ng of LASSO probe β -Actin and LASSO probe GAPDH (pre- LASSO probe sequences available in Supplementary Table 1 ). Hybridization and capture were performed as described above and the sizes of the captured ORFs were verified by gel electrophoresis. To confirm the identities of the captured ORFs, the captured GAPDH and β -Actin were cloned into a pMiniT vector (NEB) using a NEB PCR cloning kit. The purified colony PCR products from the single transformants containing or RPLP0 in pMiniT were analyzed by Sanger sequencing.
2.7. Post-capture PCR
The captured ORFs were amplified using 10 μl of the capture reaction containing DNA circles in 50 μ l of PCR master mix composed of 5 ul of 10 x Klentaq DNA Polymerase Buffer, 50 μl of Omni Klentaq LA, 200 μ M of dNTPs and 0.4 μ M of primers CaptF and CaptR. The PCR thermal profile was initiated for 3 min at 95 °C, followed by 25 cycles of 20 s at 98 °C, 15 s at 60 °C and 2 min s at 72 °C, and then 4 min at 72 °C. To visualize the amplicons derived from the circles, 3 μ l of the PCR product was loaded in a 1.1% agarose gel containing Ethidium Bromide (Sigma) (0.2 μ g ml−1).