A photodeprotection rate of 95% is generally sufficient for hybridization-based assays, but the exposure can be, e.g. doubled, to reach a photodeprotection rate of 99.8% for deletion-sensitive syntheses, such as aptamers . After all the synthesis cycles, the chemical deprotection step (vide supra) yields surface-tethered DNA. An optional capping step, to prevent the growth of oligonucleotide strands after failed coupling reactions, can also be performed, although for on-array assays it is probably not necessary in most cases since there can be no purification step to remove truncated sequences. The standard acetic anhydride capping reaction, a 1:1 mix of tertbutylphenoxyacetyl acetic anhydride (Tac 2 O) in tetrahydrofuran (Cap A) and 10% N-methylimidazole in tetrahydrofuran/pyridine (8:1) (Cap B) can be performed in photolithographic synthesis , but capping with 5'-DMT-dT phosphoramidite (or Unicap phosphoramidite ) immediately after the coupling reaction is much more efficient . When cleavage of the nucleic acid library from the surface is desired, the final coupling of the linker to the surface can be performed with a base cleavable linker . After synthesis, a 1:1 mix of ethylenediamine in toluene is used for deprotection, which additionally cleaves the library from the surface. Since the library is insoluble in the deprotection solution, it remains weakly bound to the surface, and after a further wash with acetonitrile to remove the ethylenediamine, it can be removed with a small amount of water. Because of the similarity between standard column-based synthesis and photolithographic synthesis, nucleic acid synthesizers can be repurposed with minimal modifications. The modifications are: the replacement of argon with helium as gas input (to prevent bubble formation-and hence light scattering-in the flow cell), replacement of Luer column connections with threaded connectors (to connect with small inner diameter tubing to/from the distal flow cell), and introduction of DMSO (with 1% imidazole) as exposure solvent. The use of DMSO requires operation above 20 °C to prevent flow restriction due to DMSO freezing or high viscosity near its freezing temperature. The chemistry is directed by two files, a sequence file and a protocol file. The sequence file contains the distinct chemical cycles necessary for any given synthesis as well as cleaning/priming cycles to remove dust from the flow cell. A typical sequence supersequence file would have the form: ssTTTTTACGTACGTACGT…, with the symbol "s" used for cleaning. The symbols A, C, G and T would generally be used to represent the chemical cycles needed to synthesize the corresponding DNA nucleobases, but specifically instruct the nucleic acid synthesizer, in our case an Expedite 8909, to carry out a preprogrammed set of user-defined fluidics instructions contained in the protocol file. The protocols for photolithographic synthesis are similar to those used in standard column-based DNA synthesis, as detailed in Table , which shows a generic cycle. In the photolithographic approach, synthesis starts with a coupling reaction rather than a deblocking step because the surface is functionalized with reactive groups, typically hydroxyl, rather than the DMTr-protected initial nucleotide in a standard solid support. In the deblocking cycle for photolithographic synthesis, the deblock solution is replaced with an exposure solvent, which needs to fill the flow cell prior to the photoexposure start, and also contains two trigger commands to start and end the exposure. Another difference is the use of a drying step after the coupling reaction, and the typical omission of a capping step, both as described above. A final key difference is the use of additional pulses of activator to push the monomer-activator mix to the more distal flow cell. These pushing pulses are also used in the oxidation and deblocking cycles. The number of these pushing pulses, as well as the overall consumption of solvents and reagents, is reduced though the use of small ID tubing to connect the nucleic acid synthesizer to the flow cell, and needs to be determined in each MAS setup to ensure that reagents reach the flow cell at the expected time in each synthesis cycle. Extensive additional information about the chemistry and fluidics is available in the supplementary material.