Cfx96 optical system software

RNA was extracted from cells or tissues by Trizol (Invitrogen, 15596018) and treated with RQ1 DNase I (Promega). The patient postmortem sample information is listed in Supplementary Table 2. cDNA was synthesized by High-capacity cDNA reverse transcription kit (Applied Biosystems, 4368813) using random hexamers. Regular PCR reaction was performed using LiTaq DNA polymerase (Lifesct, M0024). Normal qPCR reactions were performed with three biological replicates for each group and two technical replicates using the Power Up SYBR Green Master Mix (Applied Biosystems, A25776). The data were analyzed using the CFX96 optical system software (Bio-Rad; ver. 1.1). Expression values were normalized to GAPDH mRNA. The endogenous C9ORF72 intron 1 and mRNA levels were detected by TaqMan gene expression assay using TaqMan fast advanced master mix (Applied Biosystems, 4444557), and normalized to β-actin mRNA. Custom primer sequences are listed in Supplementary Table 3. Human ACTB primer set (Hs.PT.39a.22214847) was purchased from Integrated DNA technologies.
For RNase R treatment, 10 µg of DNase I digested total RNA was incubated at 37 °C for 15 min with 15U RNase R (Lucigen, RNR07250) to degrade liner RNA, and subsequently purified by Quick-RNA Microprep Kit (Zymo research, R1050). One microgram of RNA before or after RNase R treatment was used as the template for cDNA synthesis.

To isolate total RNA from cells, Trizol (Invitrogen) and treatment with RQ1 DNase I (Promega) was used. For first-strand cDNA synthesis, random hexamers were used with High-capacity cDNA reverse transcription kit (Applied Biosystems).
All qRT-PCR reactions were performed with three biological replicates for each group and two technical replicates using the iQ SYBR green supermix (Bio-Rad) on the CFX96 real-time PCR detection system (Bio-Rad). The data were analyzed using the CFX96 optical system software (Bio-Rad; version 1.1). Expression values were normalized to GAPDH mRNA. Intergroup differences were assessed by two-tailed Student’s t-test. Primer sequences are listed in Supplementary Table 1.
For cell fractionation, cells were lysed in gentle lysis buffer (20 mM Tris pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.3% (v/v) NP-40). Nuclei were pelleted at 2300g for 5 min at 4 °C, and supernatant (cytosol fraction) was transferred to a new tube. The nuclei were re-suspended in gentle lysis buffer and spun down again to collect the pellet (nuclear fraction). Trizol was directly added to the two fractions for subsequent RNA extraction.