E coli clone n 29664

For LASSO probe capture optimization experiments, we used a 7,249 bp circular, single-stranded DNA isolated from the M13mp18 phage (NEB) or alternatively the double-stranded, covalently closed, circular form of DNA derived from bacteriophage M13 (NEB). For capture experiments of E.coli ORFeome by MIP or LASSO probes, total genomic DNA of the E.coli strain K12 substrain W3110, (Migula) Castellani and Chalmers (ATCC 27325) was extracted from 500 μl of LB broth (Sigma Aldrich) overnight culture using Charge Switch gDNA Mini Bacteria Kit (Life Technology). Sheared total genomic DNA of E.coli K12 was obtained by sonicating 1 μg of total DNA in a volume of 200 μl in a 1.5 ml Eppendorf tube on ice by using a Branson sonifier 450 (VWR scientific) at output control 2, duty cycle 50% for 40 sec. For the capture of the 815 bp long kanamycin resistance gene KanR2, we used total DNA of the E.coli clone n 29664 (Addgene) that contained the pET StrepII TEV LIC cloning vector harboring KanR2 gene.

The capture of the 620 bp, 1 kb, 2 kb and 4 kb target sequences located in the DNA of the phage M13 were performed with the same gap filling mix composition and the same thermal profile for hybridization and capture used for the LASSO probe libraries as described above. We used approximately 0.3 fmol of single LASSO probes, and 4 fmol of M13Mp18 dsDNA or ssDNA. The E.coli k12 total genomic DNA background was 10 pM (500 ng DNA in15 μl capture volume).
For the LASSO probe sensitivity test, E. coli K12 total genomic DNA background was ~500 fM (25 ng in15 μl capture volume). The concentration of M13Mp18 dsDNA was ~500 fM (0.03 ng in 15 μl). The serial dilution concentration of the LASSO 1kB probe were 500 pM, 50 pM, 5 pM and 500 fM.
Capture of KanR2 gene was performed by using 20 ng of total genomic DNA of E.coli clone n 29664 (Addgene) 3 fmol of LASSO probe KnaR2 (pre-LASSO KnaR2 assembled with 442 bp Long Adapter). Capture was performed using the same gap filling mix and thermal profile used for the LASSO probe pool. The DNA sequences of single pre-LASSO probes are in Supplementary Table 3.