GSE211491
clampFISH 2.0 enables rapid, scalable amplified RNA detection in situ
We developed an amplified single-molecule RNA FISH method called clampFISH 2.0. To validate its performance, we used clampFISH 2.0 on two cell lines, WM989 A6-G3 cells and WM989 A6-G3 cells grown in vemurafenib, and across 10 genes, compared clampFISH 2.0 spot counts to the FPKM values from bulk RNA sequencing. Overall design: Resistant colonies were isolated from a plate of WM989 A6-G3 cells which were treated with 1µM vemurafenib. Bulk RNA sequencing was performed as described in Goyal et al. 2021 (https://doi.org/10.1101/2021.12.08.471833) for each resistant colony as well as drug-naive WM989 A6-G3 cells. For this analysis, a single resistant colony was included for comparison to the drug-naive WM989 A6-G3 cells. Briefly, we conducted standard bulk paired-end (37:8:8:38) RNA sequencing using RNeasy Micro (Qiagen, 74004) for RNA extraction, NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB E7490L), NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770L), NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) oligos (NEB, E7600S), and an Illumina NextSeq 550 75 cycle high-output kit (Illumina, 20024906), as previously described (Mellis et al. 2021; Shaffer et al. 2017). Prior to extraction and library preparation, the samples were randomized to avoid any experimental and human biases. We aligned RNA-seq reads to the human genome (hg19) with STAR v2.5.2a and counted uniquely mapping reads with HTSeq v0.6.1 (Dobin et al. 2013; Mellis et al. 2021; Shaffer et al. 2017) and outputs count matrix. The counts matrix was used to obtain tpm and other normalized values for each gene using scripts provided at: https://github.com/arjunrajlaboratory/RajLabSeqTools/tree/master/LocalComputerScripts