Organoid Isolation and qRT-PCR Analysis Protocol

Remove the coverslip of the eCHIP and the hydrogel-cell complex from the wells using ophthalmic forceps. Carefully transfer all wells with the same treatment into a 15 ml centrifuge tube to maintain their integrity.

To remove the organoids complex from the hydrogel, use Cell Recovery Solution (Corning, 354253) on ice for 1 hour. Then, centrifuge at 600g for 5 minutes. Resuspend the organoid precipitates with Advanced DMEM/F12 and centrifuge again under the same conditions to wash away residual hydrogel and cell recovery solution. Discard the supernatant as carefully as possible.

For qRT-PCR experiments, RNA extraction was performed using the RNA-Quick Purification Kit (RN001, ES Science, China) following the manufacturer's instructions. The concentration and purity of total RNA were determined using Nanodrop-2000. Reverse transcription was performed using the HiScript II 1st Strand cDNA Synthesis Kit (R211-01, Vazyme Biotech, China) according to the manufacturer's protocol.

qRT-PCR was performed using the BeyoFast™ SYBR Green qPCR Mix (D7260, Beyotime Biotech, China) on a CFX96 thermal cycler (BioRad, Hercules, CA, USA) following a preset procedure:

• Step 1: Denaturation at 95℃ for 3 minutes.
• Step 2: 40 cycles of 95℃ for 10 seconds, 56℃ for 15 seconds, and 72℃ for 15 seconds.

The reference gene used was 18S.