Nanodrop 8000

Skeletal muscle, spinal cord, and liver were harvested from treated or untreated SMN 2B/- or 2B/+ mice. Samples for RNA isolation were frozen in RNALater (Qiagen). Sample for protein analysis were flash frozen in liquid N₂. Spinal cord and liver RNA was extracted and purified according to the protocol for the RNAeasy (Qiagen). Skeletal muscle RNA was extracted and purified using the Fibrous Tissue RNAeasy mini kit. All RNA was quantified using the NanoDrop 8000 UV/Vis spectrophotometer and large RNA quality was confirmed using Bioanalyzer (Agilent). RNA analysis by ddPCR was performed as described above.

Cells were plated at a density of 4X10⁵/ml cells and were treated with either CLF alone, Ixa alone, or combination. 24hrs after incubation, high-quality RNA was isolated using QIAshredder and RNAeasy mini kit (Qiagen). RNA concentration and integrity were determined using the Nanodrop-8000 and Agilent 2100 Bioanalyzer and stored at -80°C. RNA integrity number (RIN) threshold of 8 was used for RNA-seq analysis. RNA-seq libraries were constructed using Illumina TruSeq RNA sample preparation kit v2. Libraries were then size-selected to generate inserts of ~200bp. Next-generation RNA sequencing was performed on Illumina’s NovaSeq platform using 150bp paired-end protocol with a depth of >20million reads-per-sample.

HMCLs were plated at a density of 4 × 10⁵ cells per mL, and 0.5 μM of 17-AAG was added as a single agent or in combination with 15 nM of Ixazomib. Baseline (untreated) and post-treatment (treated) cells were collected 24 h post-treatment. High-quality RNA was extracted using QIAshredder and RNeasy kit (Qiagen). RNA concentration and integrity were assessed using Nanodrop-8000 and Agilent 2100 Bioanalyzer and stored at −80 °C. An RNA integrity number threshold of eight was applied, and RNA-seq libraries were constructed using Illumina TruSeq RNA Sample Preparation kit v2.
NGS Libraries were size-selected, and RNA sequencing (RNAseq) was performed on Illumina’s NovaSeq platform using 150 bp paired-end protocol with a depth of >20million reads per sample.

Our study sampled a single site of the primary tumor. All DNA and RNA were isolated using a co-isolation protocol in which nucleic acids were isolated from the same cryopulverized aliquot that was used for both proteomics and genomics. Tumor samples were from surgical resections due to the requirement to process a minimum of 125mg of tumor issue and 50mg of adjacent normal tissue. RNA and DNA were extracted from tumor and adjacent normal specimens using QIAGEN’s QIAsymphony DNA Mini Kit and QIAsymphony RNA Kit. Genomic DNA was also isolated from peripheral blood (3-5 mL) to serve as matched benign reference material. The Qubit dsDNA BR Assay Kit was used with the Qubit® 2.0 Fluorometer to determine the concentration of dsDNA in an aqueous solution. A sample that passed quality control and produced sufficient DNA yield various genomic assays was sent for genomic characterization. RNA quality was quantified using the NanoDrop 8000 and quality was assessed using Agilent Bioanalyzer. A sample that passed RNA quality control and had a minimum RNA integrity Number (RIN) score of 8 was subjected to RNA sequencing.

High‐quality RNA was extracted from peripheral blood of AH and his mother and father collected in PAXgene blood RNA tubes PreAnalytiX GmbH, Switzerland using PAXgene blood RNA kit PreAnalytiX GmbH, Switzerland. RNA concentration and integrity (RNA integrity number/RIN) were evaluated using the Nanodrop‐8000 and Agilent 2100 Bioanalyzer and stored at −80°C. Dual indexed TruSeq stranded RNA libraries were prepared using Illumina TruSeq RNA sample Preparation kit v2. The libraries were size selected to generate inserts of ~200 bp, pooled and RNA with RIN>8 were sequenced on llumina's HiSeq 2500 high‐output 50 next‐generation high‐throughput sequencing system using 50 bp paired‐end run using v4 chemistry with depth of >20 million reads‐per‐sample. Average quality scores for all libraries were above Q30. Data were normalized and FPKM values were used in further analysis using a combination of Galaxy data analysis software and Partek Genomics Suite. Gene expression profiling (GEP) data was then used to identify highly significant “EHMT1‐associated genes” (details in Results).

PBMCs were prepared for gene expression analysis by removing the media from the cells, rinsing with room temperature phosphate buffered solution pH 7.4 (Quality Biologicals Inc., Gaithersburg, MD) and lysing the cells via resuspension in Buffer RLT (Qiagen, Valencia, CA). The lysed cell mixture was stored at −80 °C. Total RNA was isolated from PBMCs that underwent different storage conditions using Qiagen RNeasy kit by following manufacturer’s protocol. Total RNA was quantitated and qualified in Nanodrop 8000 and Agilent Bioanalyzer RNA Nano 6000 chip before performing Affymetrix GeneChip assays. Affymetrix Human GeneChip U133 Plus 2.0 Array (Affymetrix, Sacramento, CA) was used and labeling assay was performed by following manufacturer’s protocol.
Microarray data was normalized and analyzed using Partek Pro (St. Louis, MO). Genes were selected by 3-way ANOVA statistical analysis and correlated with cell viability flow cytometry data. The selection criteria are P-value less than 0.05 and absolute fold change more than 3. The correlation selection criteria are p-value is less than 0.05 and inter-quartile value is more than 1.4. Selected genes were normalized by medium shift before hierarchy clustering. Biological function annotation was done on in-house software DAVID [29 (link)] (http://david.abcc.ncifcrf.gov/) and Ingenuity IPA software (Qiagen, Germantown, MD).