RNA seqFISH+ Supplementary Protocol

Long Cai,Chee Huat (Linus) Eng

doi:10.1038/protex.2019.019

Abstract

RNA seqFISH+ Supplementary Protocol

Highlights

This supplementary protocol describes the sample preparation for the experiments in RNA seqFISH+ in detecting 10,000 or more genes in both cell culture and tissue slice
For cell culture in seqFISH+ experiment, the hybridization is typically 3648 hours which we found 48 hours sometimes yield slightly brighter signals
If it is too difficult to remove the coverslips on top of the cell/ tissue slice, place 1 mL of 40% wash buffer \(40% formamide, 2x SSC, 0.1% Triton-X 100) at room temperature covering the entire coverslip and samples for 15 minutes or more

Summary

Introduction

This supplementary protocol describes the sample preparation for the experiments in RNA seqFISH+ in detecting 10,000 or more genes in both cell culture and tissue slice. The steps include coverslips functionalization, primary probes hybridization, washing, hydrogel embedding, tissue clearing and restabilization of the samples for prolonged imaging for both barcoded and non-barcoded experiments as demonstrated in RNA seqFISH+ manuscript. 3. On the day of primary probes hybridization, we first permeabilize the tissue slices at 4°C for >1 hour by directly placing the coverslip in a 50mL Falcon tube full of 70% ethanol.

Results

Conclusion