15 µl of cell extract was incubated with 500 µl ß-galactosidase assay buffer (100 mM HEPES pH 7.3 KOH, 150 mM NaCl, 4.5 mM Aspartate (hemi-Mg salt), 1 % w/v bovine serum albumin, 0.05 % v/v Tween 20, 1.6 mM chlorphenol red- ß-D-galactopyranosid) until colour change. The reaction was then stopped by adding 250 µl 3 mM ZnCl2 and absorbance was measured at 578 nm. ß-galactosidase values were calculated using the following formula: ß-gal units = 1000 x OD578 nm / t x OD595 nm where t is the time of incubation of the ß-galactosidase assay buffer with the sample before addition of ZnCl2. To determine efficiency of cell lysis, protein content was measured using a Bradford assay. 1 ml 1:5 diluted Bio-Rad Protein assay solution (BioRad) was incubated with 10 µl sample. Following short incubation time absorbance was measured at 595 nm.
LAM-PCR for Illumina sequencing
Linear amplification. Linear PCR reactions were done in 20 mM Tris pH 8.4, 50 mM KCl, 1.5 mM MgCl2, 0.2 mM dNTP, 0.02 pM biotinylated primer LAM-SB/L-50Bio, 2.5 U Taq-Polymerase. PCR program: 94 °C 4 min, 50 cycles of 94 °C 40 sec, ramp to 54 °C 1 °C/sec, 54 °C 30 sec, 72 °C 1 min. After one round of linear PCR, an extra 0.5 µl of Taq Polymerase was added and samples were subjected to a second round of linear PCR.
Magnetic capture. Biotinylated linear PCR products were captured by streptavidin coupled beads (Dynabeads kilobaseBINDER Kit, Invitrogen). Upon exposure to a magnet DNA coupled to the beads was separated from supernatant. Magnetic capture was done according to manufacturer´s protocol.
Second strand synthesis. Linear PCR products were converted into dsDNA. The second strand synthesis reaction was performed using 2 µl 10 x Hexanucleotide mix (Roche), 0.25 mM dNTP, 2 U Klenow fragment (Fermentas), x µl H2O in 20 µl on linear DNA coupled to magnetic beads.
End repair. Possible overhangs of dsDNA created by second strand synthesis were blunt-ended and 5´-ends phosphorylated using the End It DNA End-Repair Kit (Epicentre Biotechnologies) according manufacturer´s instructions.
Adding A´-overhangs. A´-overhangs were added to the 3´-ends of DNA molecules. Magnetic beads were incubated in 5 µl NEB2 buffer, 0.2 mM dATP, 1 µl Klenow fragment Exo (-) (New England Biolabs) and 43 µl H2O for 30 min at 37 °C following 20 min at 75 °C for heat inactivation.
Linker ligation. A double stranded sonic linker with T´-overhangs was ligated to DNA coupled to beads using the Fast-Link DNA Ligation Kit (Epicenter Biotechnologies): 1 µl 10x ligation buffer, 1 µl 10 mM ATP, 1 µl Fast-Link ligase, 1 µl 50 pmol/µl sonic linker. Ligation reaction was carried out at 4 °C overnight.
Nested PCR. After ligation DNA plus linker coupled to beads was resuspended in 10 µl TE. 2 µl of bead suspension, 1 µl 10 mM dNTP, 1 µl 10 pmol/µl linker primer, 1 µl 10 pmol/µl LAM SBL-20 hmr, 2.5 U Taq polymerase, 3 µl 25 mM MgCl2, 5 µl 10 x Taq buffer and H2O to fill up the reaction to 50 µl were PCR amplified using the following PCR program: 94 °C 2 min followed by 35 cycles of 94 °C 30 sec, ramp to 55 °C 1 °C/sec, 55 °C 20 sec, 72 °C 30 sec ended by 72 °C 5min. A nested PCR was performed with 1 µl of PCR 1, 1 µl 10 mM dNTP, 1 µl 10 pmol/µl nested primer, 1 µl 10 pmol/µl primer SB III barcode_OVH, 2.5 U Taq polymerase, 3 µl 25 mM MgCl2, 5 µl 10 x Taq buffer and H2O to fill up the reaction to 50 µl were PCR amplified using the following PCR program: 94 °C 3 min followed by 35 cycles of 94 °C 30 sec, ramp to 51 °C 1 °C/sec, 51 °C 20 sec, 72 °C 30 sec ended by 72 °C 5 min. Barcoded primers annealed at the end of the SB transposon. They were identical except for four bases within the primer. All PCR reactions ascribing to one transposase (fused, mixed or unfused) were done with the same barcoded primer, so that transposon integrations mapped from PCR products could be traced back to corresponding transposases. A third PCR was performed to add overhangs necessary for recognition by the Genome Analyzer IIx (Illumina). 1 µl of PCR 2, 1 µl 10 mM dNTP, 1 µl 10 pmol/µl Illumina Pr1, 1 µl 10 pmol/µl Illumina Pr2, 0.5 µl Phusion polymerase, 10 µl Phusion buffer and H2O to fill up the reaction to 50 µl were PCR-amplified using the following PCR program: 98 °C 30 sec followed by 14 cycles of 98 °C 10 sec, 65 °C 30 sec, 72 °C 30 sec ended by 72 °C 5 min. PCR reactions were run on an agarose gel and verified for product sizes ranging from 100 bp to 500 bp. Equal amounts for all samples with differing barcodes were mixed together and loaded to a Genome Analyzer IIx (Illumina) analyzer.