Stranded mrna prep kit

Library preparation and sequencing were conducted at the Iowa Institute of Human Genetics Genomics Sequencing Division core facility, University of Iowa. Briefly, an Illumina Stranded mRNA Prep kit was used to isolate oligo-dT purified polyadenylated RNA. The samples were then reverse transcribed to create cDNA. The cDNA was fragmented, blunt-ended, and ligated to indexed adaptors. Following quantification of the cDNA generated for the library, the samples were clustered and loaded equally over two lanes on an SP flow cell on an Illumina NovaSeq 6000 Sequencing system for 50bp paired end reads.

The NucleoSpin RNA Mini kit (Macherey-Nagel, Düren, Germany) was used to extract total RNA and RNA integrity numbers (RIN) were measured using the Agilent 4200 Tapestation (Santa Clara, United States, cat. G2991BA). RIN values of samples considered within this study ranged from 9.3–10. No significant differences in RIN values were observed between the two phenotypes. Libraries preparation was carried out using the Illumina Stranded mRNA Prep kit. Sequencing of RNA libraries was performed in accordance with standard Illumina protocols within the Genomics Core Facility of the Center for Advanced Genomics Technology (Icahn School of Medicine at Mount Sinai, New York). Libraries were pooled and 100 bp, paired-end reads were sequenced on a NovaSeq 6000. FASTQ files were aligned to GENCODE v29 [23 (link)] using STAR (v2.6.1d) [24 (link)] and RSEM (v1.3.1) [25 (link)].

Colistin-resistant E. coli B2 was grown to the exponential phase, and bacteria were cultured with colistin alone or in combination with DSF (n = 3 biologically independent samples). After incubation for 4 h, cells were collected, and total RNA of bacteria was extracted using the TRIzol reagent (Invitrogen). Next, the RNA was quantified by using a Nanodrop spectrophotometer (Thermo Scientific, MA, USA), followed by library construction, sequencing and bioinformatics analysis. The RNA-seq library was constructed using the Illumina® Stranded mRNA Prep kit (San Diego, CA). Briefly, all mRNAs were broken into short (200 nt) fragments by adding fragmentation buffer. Then, double-stranded cDNA was synthesized with random hexamer primers (Illumina) and subjected to end-repair, phosphorylation and adenine addition according to Illumina’s library construction protocol. Paired-end RNA-seq library was sequenced with the Illumina Novaseq 6000 (Illumina Inc., San Diego, CA, USA). Subsequently, the data generated from Illumina platform were used for the following bioinformatics analysis. All of the analyses were performed using the free online platform of Majorbio Cloud Platform (www.majorbio.com). The DESeq2 package was used for the differential expression analysis. Functional annotation was performed with GO and KEGG database.

RNA for cell lines was extracted with an RNAeasy Kit (Qiagen Cat# 74104), while RNA from primary cells was extracted with the Arcturus™ PicoPure™ RNA Isolation Kit (Thermo Fisher Scientific Cat# KIT0204), with DNA removed using the DNase Set‐RNase free (Qiagen Cat# 79254) following manufacturer instructions. Libraries were prepared with the Stranded mRNA Prep kit (Illumina). Samples were sequenced on Illumina NextSeq 500 using the High Output 75 cycles kit (2 × 36 cycles, paired end reads, dual index; Illumina) to obtain minimum 20 million reads per sample. Subsequently, FastQ files were generated using Illumina's bcl2fastq (v. 2.20.0.422). The sequencing data has been deposited on GEO with accession GSE226887. QC was conducted using fastqc v0.11.8, reads trimmed using trimgalore v0.4.4. Trimmed reads were then aligned to GRCh38 using Hisat2 v2.1.0 and sorted into bam files using samtools (v1.15.1.5). The bam files for two independent runs of each samples were merged using samtools (v1.15.1.5). Read count extraction was performed using featureCounts function in Subread package (v2.0.1) and resulting featureCounts analysed using DESEQ2 (v1.34.0). GSEA (version 4.1) was used on pre‐ranked lists (ranked by pi score that was computed by multiplying log₂ fold change by −log₁₀ (corrected P‐value)).

Total RNA was isolated using Trizol (Invitrogen). RNA-seq libraries were constructed using Illumina Stranded mRNA Prep kit as described in the manual and subjected to 150-bp single end sequencing on HiSeq 2500. STAR aligner (version 2.5.2b) was used to align reads to the mouse genome (GRCm38.p5) using annotation from Ensembl release 99 with reads-per-gene count enabled as described in the manual. The DEseq2 package was for differential expression analysis using R statistical software (version 3.6.0). For expression level visualizations, we used regularized log transformation of the count data, as described in the package vignette. To test whether there were any sets of related genes in the list of differentially expressed genes we used the gage-package in combination with gene sets from MSigDB. Correction for multiple testing was performed using false discovery rate with the Benjamini-Hochberg method.

RNAseq was performed on RNA isolated from neonatal female cerebral cortices 5 days after IMQ administration (GD19). Total RNA was extracted using the RNeasy Plus Mini Kit (#74134, Qiagen, Germantown, MD, USA) according to the manufacturer’s instructions. Genomic DNA was depleted with DNAse1 treatment. RNA quality check, library preparation, and sequencing were performed by Princess Margaret Genomics Centre (Toronto, ON, Canada). Briefly, RNA quality was assessed using an Agilent 2100 Bioanalyzer for an RNA integrity number (RIN) greater than 8. Stranded mRNA libraries were prepared using an Illumina Stranded mRNA Prep Kit and sequenced on an Illumina Novaseq 6000 sequencer using paired-end reads (2 × 100 bp) with a read depth of 25 million. FASTQ files were quality checked using FASTQC and processed on the Galaxy platform [22 (link)]. Reads were mapped onto the rat genome (rn6) using STAR [23 (link)] and differentially expressed genes were identified with DESeq2 [24 (link)]. Enriched genes were subjected to pathway enrichment analysis with g:Profiler [25 (link)] and cross-referenced with the Simons Foundation Autism Research Initiative (SFARI) database of genes with known associations with ASD [26 (link)].

For RNA-seq of tissues from WT, APC or APC KRAS mice, 1 µg of RNA was prepared in 50 µl. RNA-seq was performed using an Illumina TruSeq RNA sample prep kit, then run on an Illumina NextSeq using the High Output 75 cycles kit (2 × 36 cycles, paired-end reads, single index). For RNA-seq of tissues from APC mice treated with vehicle or with DZNeP, 1 µg of RNA was prepared in 25 µl. RNA-seq was performed using an Illumina Stranded mRNA Prep kit, then run on an Illumina NextSeq 500 using the High Output 75 cycles kit (2 × 36 cycles, paired-end reads, dual indexed).
The raw sequence quality was assessed using the FastQC algorithm version 0.11.8. Sequences were trimmed to remove adaptor sequences and low-quality base calls, defined by a Phred score of <20, using the Trim Galore tool version 0.6.4. The trimmed sequences were aligned to the mouse genome build GRCm38.98 using HISAT2 version 2.1.0, then raw counts per gene were determined using FeatureCounts version 1.6.4. Differential expression analysis was performed using the R package DESeq2 version 1.22.2, and PCA was performed using R base functions.