Novaseq 6000 pe150

PCR-amplified Nf1 products from the seven variable sequences were randomly sheared into 350 bp fragments through ultrasonic disruptors, then end repaired, A-tailed, and further ligated with Illumina adapters. The fragments with adapters were size-selected, PCR amplified, and purified. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina NovaSeq6000 PE150 platforms, according to effective library concentration and data amount required. Fastq files sequence quality was confirmed via FastQC. Sequences with a per base quality score over 28 were retained for downstream analysis. Phred quality scores were checked for error probability in base calling (≥ 30). Next, sequences were aligned and indexed via Galaxy workflow, BWA-MEM2. Bam and bai files were input into IGV_2.16.1 to visualize alignments. Finally, absolute max indel sizes observed via NGS were compared to the max indel sizes observed via TIDE, Synthego, and DECODR for each sample.

Total RNA was extracted from cells with Trizol reagent (Invitrogen, CA, USA). Random hexamer-primers were used to synthesise first-strand cDNA. Second-strand cDNA was synthesised using buffer, dNTPs, RNase H and DNA polymerase I. After adapter ligation and agarose gel electrophoresis, suitable fragments were selected for PCR amplification. Finally, the RNA-seq library was sequenced using an Illumina Novaseq 6000 PE150 instrument by Haplox Biotechnology Co. (ShenZhen, China).

Cells for single-cell RNA sequencing were obtained from cerebral cortex of P1 mTrappc9^+/+ and mTrappc9^m/m mice born in the same litter. The tissues were digested with Papain Dissociation System according to the manufacturer's protocol. Live cells were sorted using flow cytometry, with a minimum of 2 million cells were collected in each group. Libraries were prepared using BD Rhapsody^TM technology. Sequencing platform was Novaseq 6000 PE150 of Illumina. Raw data analysis was performed using R version 4.0.2. as described previously91 (link).

After neuronal maturation for 28 days, total RNA was collected from the DPSC-neurons using the Zymo Directzol RNA extraction kit (Zymo, Irvine, CA). Prior to sequencing, RNA was assayed for integrity and quality using the Agilent Bioanalyzer 6000 pico chip (Agilent, Santa Clara, CA). Only RNA with an RNA Integrity Number (RIN) ≥9.0 was used for RNAseq studies. Library preparation and RNAseq was performed by Novogene (NovaSeq 6000 PE150) (Sacramento, CA) using the Illumina platform and paired end reads. 20 M paired-end reads per sample were collected.

The input RNA (10 ng fragmented RNA) and total IP RNA were used as starting materials to construct libraries with SMARTer Stranded Total RNA-Seq Kit v2-Pico Input Mammalian (Takara-Clontech, 634488), according to the standard protocol. The input RNA underwent 14 PCR cycles, while the m⁶A RNA was subjected to 16 cycles. Sequencing was performed using Illumina NovaSeq 6000 PE150.

Whole exome sequencing was done using SureSelect Human All Exon V6 (Agilent Technologies) and Illumina Novaseq6000/PE150. Reads mapping and further analyses were done using Genomon pipeline (https://github.com/Genomon/genomon). The GRCh37 reference genome was used for alignment.