Rna analysis screentape

RNA was extracted from C. strossmayeri mycelium, harvested from a two-week culture grown in PDB, using the E.Z.N.A^® Fungal RNA Kit (OMEGA bio-tek). Isolated RNA was quality checked using RNA Analysis ScreenTape^® (Agilent). Approximately 500 ng of total RNA was prepared for sequencing using the Illumina TruSeq Total RNA LT Kit (Illumina). The data were processed using RTA version 1.18.64, with default filter and quality settings. The reads were demultiplexed with CASAVA 1.8.4, allowing no mismatches. This was carried out at the Bristol Genomics Facility. RNA-seq data were processed using Galaxy QC and manipulation tools to trim the sequences followed by the TopHat RNA analysis tool to map the RNA-seq reads to the assembled genomes. These data were then viewed in Artemis to evaluate relevant expression levels of genes of interest. Partek^® Genomics Suite was also used to map RNA-seq reads to assembled genomes and to genes of interest. RNA-seq data are available on the NCBI SRA database under the accessions: STUDY: PRJNA604530; SAMPLE: CBS 177.39 (SAMN13973684); EXPERIMENT: C.s (SRX7684385); RUN: AB_C_ACAGTG_L001_R1_001.fastq (SRR11032120).

All samples were lysed in Trizol (note the additional steps used for sperm described above) using phenol-chloroform separation. The aqueous phase was then processed with Zymo RNA Clean and Concentrator Kit with DNAse1 on-column treatment (Zymo Research, Irving, CA). Final sperm RNA concentrations were determined with Qubit RNA HS assay (Thermo Fisher) and RNA Analysis ScreenTape (Agilent, Santa Clara, CA) was used to confirm absence of 18S and 28S ribosomal peaks that are indicative of somatic cell contamination.

RNA libraries were prepared according to the manufacturer’s instructions for the Illumina TruSeq Stranded mRNA library prep kit (Illumina, Inc., California, USA). The libraries were sequenced with the HiSeq 2500 sequencing platform (Illumina, Inc.). The quantity and quality of RNA and library were measured respectively with RNA Analysis ScreenTape (Agilent Technologies, California, USA) and D1000 ScreenTape using 2200 TapeStation System (Agilent), respectively. The data was analyzed using several steps: For Read alignment and gene counts STAR 2.6.1d was used using the ENSEMBL hg38 reference build from Illumina iGenomes. The quality control is done using FastQC 0.11.4 and MultiQC v1.8 for summarization of the quality metrics. For the differential gene expression and normalization, the DESeq2 R package was used in combination with heatmap for the heatmaps. Significant differences between samples are expressed with False Discovery Rate (FDR) value less than 0.05. Further analysis with significantly different expression genes was performed with Metascape gene ontology (https://metascape.org/gp/index.html#/main/step1) to investigate biological gene functions and interactions.

To determine transcriptome profiles over the course of differentiation, three hiPSCs lines (ATCC, BJFF, and STAN) as biological replicates at various differentiation stages (6 mesodermal and 5 chondrogenic stages per cell line; i.e., total 33 samples) were collected for bulk RNA-seq. Cell samples were thawed on ice, and pellet samples were homogenized with zirconia beads (BioSpec Products, # 11079110zx) and a miniature bead beater. RNA was then isolated from all samples using the Total RNA Purification Kit according to the manufacture’s protocol (Norgen Biotek, #37500). RNA was eluted in 20 μl of diethylpyrocarbonate-treated water. The quality and quantity of RNA from each sample was evaluated by RNA Analysis ScreenTape (Agilent, #5067-5576) on a bioanalyzer (Agilent 4200 Tapestation). Only samples with a RIN value larger than 0.8 were submitted to the Genome Technology Access Center (GTAC sequencing core) at Washington University in St. Louis for library preparation and bulk RNA-seq. Libraries were prepared using TruSeq Stranded Total RNA with Ribo-Zero Gold kit (Illumina). Sequencing was performed on a HiSeq2500 instrument (Illumina) (1 × 50 bp reads) with a sequencing depth of 30 million reads per sample.

RNA was isolated from bacteria using TRIZOL and quantified using Qubit RNA BR Assay Kit (Life technology, Q10210). To quantify over-expressed RNA, 50 ng of total RNA was size separated using RNA ScreenTape Analysis (Agilent, 5067–5576) and quantified using TapeStation software.

The ChIP purified A/B standards were submitted to an in vitro transcription reaction, incubated overnight at 37 °C, using the MEGAscript T7 Transcription kit (ThermoFisher) according to the manufacturer’s protocol. The resulting product was then treated with Turbo DNase and the remaining RNA was purified with the Zymo RCC-25 column purification-25 kit (Zymo Research). The A/B RNA standards were quantified using Qubit RNA HS Assay on Qubit 2.0 Fluorometer (Life Technologies) and then verified on the Agilent TapeStation (Agilent Technologies) with the RNA ScreenTape Analysis (Agilent Technologies).