Microfluidic Device Design and Fabrication

Microfluidic devices were designed using AutoCAD (AutoDESK, USA), tested using COMSOL Multiphysics as well as
empirically, and fabricated using soft lithographic techniques^{22 (link)} (Supplementary Data 1). The devices were tested on a Drop-seq setup,
using bare beads (Tosoh, Japan, Cat # DTTiCZIBPBHhf-iF1YcK/" target="_blank">HW-65s) in Drop-Seq Lysis Buffer (DLB^{7 (link)}; 10 ml stock consists of 4 ml of nuclease-free H₂O, 3 ml 20% Ficoll PM−400
(Sigma, Cat # F5415-50ML), 100 μl 20% Sarkosyl (Teknova, Cat # S3377), 400 μl 0.5M
EDTA (Life Technologies), 2 ml 1M Tris pH 7.5 (Sigma), and 500 μl 1M DTT (Teknova, Cat # D9750), where the DTT
is added fresh) and 1x PBS, to optimize flow and bead occupancy parameters in drops. Droplet generation was assessed under a
microscope in real time using a fast camera (Photron, Model # SA5), and later by sampling the emulsion using a
disposable hemocytometer (Life Technologies, Cat # 22-600-100) to check droplet integrity, size, and bead occupancy.
The device design is provided as a Supplementary File 1 and Supplementary Fig. 1a. The unit in the CAD provided is 1 unit =
1 μm; channel depth on device is 75 μm.