Rta software

Total RNA was extracted using a RNeasy kit (Qiagen, USA). The RNA quality was examined using an Agilent 2100 BioAnalyzer (Agilent Technologies, USA), NanoDrop (Thermo Fisher Scientific Inc.), and 1% agarose gel. Library preparation was constructed using a NEBNext® UltraTM RNA Library Prep Kit for Illumina®. The constructed library was validated using an Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA), and quantified by Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). Sequencing was performed on the illumine HiSeq platform in a 2X150bp paired-end configuration. Base-calling is performed by Illumina RTA software. Demultiplexing is performed by Illumina bcl2fastq 2.17 software based on index information and the number of reads and quality score (Q30) were counted. Data were aligned to reference genome via software HISAT2 (v2.0.1)^{45 (link),46 (link)}. Differential expression analysis used the DESeq2 Bioconductor package. The sequencing data were submitted to the NCBI’s Gene Expression Omnibus (GSE226347).

Raw data from four flowcells of the HiS eq 2500 machines using HiSeq (v4) SBS chemistry were processed using the Illumina RTA software and CASAVA 1.8.2, and then all samples checked for standard CASAVA QC parameters (all reads pass filter). Specifically, all samples had high (>Q20) quality values at the median base, low % alignment to PhiX (<1%), and similar insert size (550 ± SD of 70 bp).

We sequenced bisulfite-converted libraries with 2×101 base pair paired-end reads on an Illumina HiSeq 2000 instrument with conventional A. thaliana DNA genomic libraries in control lanes. Each sample contained 0.1% lambda DNA as an unmethylated control. We pooled six different samples in each lane. The Illumina RTA software (version 1.13.48) performed image analysis and base calling.

The resulting high quality RNA was employed for the generation of a cDNA library through TruSeq RNA Sample Preparation Kit (Illumina Inc.). The purified cDNA library was used for cluster generation on the Illumina Cluster Station and then sequenced on Illumina HiSeq 2000 following vendor instruction. A paired-end sequencing run with 100 nt read length for each read was performed for RNA-Seq. Raw sequencing intensities were then extracted and the bases were called using Illumina RTA software, followed by sequence quality filtering. The extracted sequencing reads were saved as a pair of fastq files for the first and second read, respectively.

Ten cortical neuron cultures (1.2 × 10⁶ for each sample) were collected from 5 WT mice and 5 Df16(A)^+/− mice at DIV7, all E17.5 male embryos. Total RNA was isolated from the cortical neurons using miRNeasy kit (Qiagen, USA) according to the instructions of the manufacturer. RNA was suspended in RNase-free water. The concentration and purity of each sample was determined by spectrophotometer (ND1000; Nanodrop, USA) and confirmed by Microchip Gel Electrophoresis (Agilent, USA) using Agilent 2100 Bioanalyzer Chip according to the instructions of the manufactures. A poly-A pull-down step was performed to enrich mRNAs from total RNA samples (200 ng to 1 µg per sample, RIN > 8 required) and proceeded on library preparation by using Illumina TruSeq RNA prep kit. Libraries were then sequenced using Illumina HiSeq2000 at Columbia Genome Center. Multiplex samples wit unique barcodes were mixed in each lane, which yields targeted number of single-end 100 bp reads for each sample, as a fraction of 180 million reads for the whole lane. RTA software (Illumina, USA) was used for base calling and bcl2fastq (version 1.8.4) for converting BCL to fastq format, coupled with adaptor trimming. The reads were mapped to a reference genome (Mouse: UCSC/mm9) using Tophat^{20 (link)} (version 2.0.4) with four mismatches (--read-mismatches = 4) and 10 maximum multiple hits (--max-multihits = 10).