T4 ligase buffer 10x

The second round of barcoding was carried out by ligation reaction (Supplementary Table T3). A new 96well plate (Round-2) was prepared on ice with 10 μL/well of barcodes from the equivalent positions in the previously annealed WD-2 plate. Then, 2 mL of ligation mix (500 μL of T4 Ligase Buffer 10x (NEB), 100 μL of T4 DNA Ligase (400 U/μL, NEB) and 1500 μL of Nuclease-free water) was added to the 2 mL of cells in 1x NEB buffer 3.1 and mixed thoroughly into a disposable basin. We added 40 μL/well of this ligation mix (including cells) to the Round-2 plate and covered it with an adhesive PCR plate seal. The plate was incubated in a thermocycler for 30 min at 37ºC. To block Linker_1 after incubation, a blocking solution was prepared with 264 μL of Blocker_1 (26.4 μM final concentration), 250 μL of T4 Ligase Buffer 10x (NEB) and 486 μL of Nuclease-free water. After incubation, the cover of the Round-2 plate was removed and 10 μL of blocking solution were added to each well, giving a final volume of 60 μL/well. The plate was sealed again, with a new adhesive cover, and incubated for another 30 min at 37ºC to bind Linker_1 and Blocker_1 oligos.