New technology maps where and how cells read their genome nature
Chrome photomasks were purchased from Front Range Photomasks, with a channel width of either 20 or 50 μm. The moulds for polydimethylsiloxane microfluidic devices were fabricated using standard photolithography. The manufacturer’s guidelines were followed to spin-coat SU-8-negative photoresist onto a silicon wafer . The heights of the features were about 20 and 50 μm for 20- and 50-μm-wide devices, respectively. PDMS microfluidic devices were fabricated using the SU-8 moulds.
For first barcode in situ ligation, the first PDMS chip was covered to the tissue region of interest. For alignment purposes, a 10× objective was used to take a brightfield image. The PDMS device and tissue slide were clamped tightly with a home-made acrylic clamp. Barcode A was first annealed with ligation linker 1, 10 μl of 100 μM ligation linker, 10 μl of 100 μM each barcode A and 20 μl of 2× annealing buffer and mixed well.
For DNA and cDNA separation, the lysate was purified with Zymo DNA Clean & Concentrator-5 and eluted to 100 μl of RNase-free water. The 1× B&W buffer with 0.05% Tween-20 was used to wash 40 μl of Dynabeads MyOne Streptavidin C1 beads three times. Then, 100 μl of 2× B&W buffer with 2.5 μl of SUPERase In RNase inhibitor was used to resuspend the beads, which were then mixed with the lysate and allowed to bind at room temperature for 1 h with agitation.
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