Methods for making flow cells

A method, comprising:

• imprinting a resin layer of a multi-layer stack to form a multi-height convex region including a first region with a first height and a second region with a second height that is smaller than the first height, wherein the multi-layer stack includes the resin layer over a sacrificial layer over a transparent layer over at least one additional layer selected from the group consisting of a base support and a hydrophobic layer over the base support;
• selectively etching portions of the multi-layer stack around the multi-height convex region to expose the at least one additional layer;
• selectively etching the multi-height convex region to remove the resin layer and a portion of the sacrificial layer underlying the second region of the multi-height convex region, thereby forming a protrusion over the base support, and exposing a first portion of the transparent layer, the protrusion including at least the transparent layer and being a different material than the base support;
• applying a first functionalized layer over the protrusion, including over the first portion of the transparent layer, and over a remaining portion of the sacrificial layer;
• lifting off the remaining portion of the sacrificial layer and the first functionalized layer thereon, thereby exposing a second portion of the transparent layer and whereby the first functionalized layer remains over a first portion of the protrusion;
• applying a second functionalized layer over the second portion of the transparent layer, and thus over a second portion of the protrusion, wherein the first and second portions are directly adjacent to each other;
• attaching a first primer set to the first functionalized layer, wherein the first primer set includes an un-cleavable first primer and a cleavable second primer;
• and
• attaching a second primer set to the second functionalized layer, wherein the second primer set includes a cleavable first primer and an un-cleavable second primer;
• wherein:
• the cleavable first primer includes a first nucleotide sequence and a first cleavage site;
• the un-cleavable first primer includes the first nucleotide sequence without the first cleavage site;
• the cleavable second primer includes a second nucleotide sequence and a second cleavage site;
• the un-cleavable second primer includes the second nucleotide sequence without the second cleavage site;
• the first cleavage site is the same as or different than the second cleavage site;
• and
• the first and second nucleotide sequences are different.