Nextseq 550 sequencing system

The total RNA extraction of AML-12 cells was performed using a TRIzol™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA), following the protocol described by the manufacturer. The concentration was analyzed using a Qubit™ RNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) in a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The purity and integrity of the RNA samples were determined in a Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA) using an Agilent RNA 6000 Pico Kit (Agilent Technologies, Santa Clara, CA, USA).
Libraries were generated using a TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA). Libraries were quantified using real-time quantitative PCR in addition to the high sensitivity DNA kit and the Agilent 2100 Bioanalyzer System (Agilent Technologies, Santa Clara, CA, USA). Barcoded libraries had their cDNA concentrations adjusted for 4 nM. Sample sequencing was performed using a NextSeq^® 500/550 High Output Kit v2 (75 cycles) (Illumina, San Diego, CA, USA) in a NextSeq^® 550 Sequencing System (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The data were pre-processed using the standard Illumina processing pipeline to segregate multiplexed reads.

CSF was collected via lumbar puncture (LPs), which was performed on all patients between 1 and 4 h at baseline and 12 months (end of treatment) with placebo or nilotinib. Cell-free total RNA was isolated from 200 µl of CSF using the Qiagen miRNAeasy extraction kit (Qiagen, 217,184) and CSF microRNA sequencing was performed as we previously explained [32 (link)]. Samples were normalized to an input volume of 5 μl RNA eluate to prepare miRNAseq libraries using Qiagen QiaSeq miRNA-seq library preparation kit (Qiagen, 331,502). Next-generation sequencing (NGS) was performed on a NextSeq 550 Sequencing System (Illumina) using single-end (SE) 1 × 75 base pairs (bp) sequencing to a depth of 25 million raw reads per sample.

Library preparation was performed by amplification and capture using the NGS gene panel detection library construction kit (Shanghai yuanqi Bio‐pharmaceutical Technology Co., Ltd.) and PE150 sequencing on Nextseq 550 Sequencing System (Illumina). The primary data were aligned to the human reference genome at NCBI, Clinvar, dbSNP (V138), COSMIC, and Human Genome database (HG19) with the determination of point mutations (SNV), insertions and deletions (INDEL), and pathogenic mutations.

Pet1 neurons from Pet1-EYFP embryos were flow sorted at E14.5 into PBS/10% FBS. FACS-purified neuron viability was confirmed to be >80% by staining a portion of the cells with Trypan blue and counting the number of live cells on a hemocytometer. Single cells were loaded in the 10× Single Cell 3’ Chip at a concentration of 1000 cells/μL. Library was prepared according to 10× Genomics Single Cell Protocols Cell Preparation Guide on the 10× Genomics Chromium controller and sequenced to a depth of over 80,000 reads per cell for ~2700 cells on Illumina NextSeq 550 Sequencing System.

CSF (200 µL) was used to isolate cell-free total RNA using the Qiagen miRNAeasy serum/plasma extraction kit (Qiagen, 217184) and CSF microRNA sequencing was performed as we previously explained [53 (link)]. Qiagen QiaSeq miRNA-seq library preparation kit (Qiagen, 331502 was used to normalize samples to an input volume of 5 µL RNA eluate and obtain miRNAseq libraries.) NextSeq 550 Sequencing System (Illumina) using single-end (SE) 1 × 75 base pairs (bp) sequencing to a depth of 25 million raw reads per sample was used to perform unbiased Next-generation sequencing (NGS). miRNA quantification was performed via uploading of FASTQ files to the online Qiagen Data Analysis Center. The unique molecular index (UMI) counts were calculated, and primary miRNA mapping was performed using a human-specific miRBase mature database. In the primary QIAseq quantification step, adaptor sequences from the library preparation process and any low-quality bases were removed. UMI counts for each miRNA were used for differential expression analysis.

PolyA mRNA was isolated from the frozen cell samples using the Dynabeads mRNA direct kit (Thermo fisher 61011). Subsequently, complementary DNA (cDNA) was prepared using the method described in Picelli et al. (54 (link)). cDNA integrity and concentration were assessed using a High Sensitivity D5000 ScreenTape (Agilent 5067-5592). A total of ∼500 pg of cDNA was used as the input to make sequencing libraries with the Illumina Nextera XT DNA Library Preparation Kit (FC-131-1096) with nine PCR cycles. Final libraries were quantified on a High Sensitivity D1000 ScreenTape on the TapeStation (5067-5584).
Libraries were run on the Illumina NextSeq 550 sequencing system. Reads were aligned to the dm6 version of the Drosophila genome using STAR (55 (link)). PCR duplicates were removed using Picard Tools (Picard Toolkit 2019. Broad Institute, GitHub Repository, https://broadinstitute.github.io/picard/; Broad Institute). Differential expression analysis between nSyb neurons and clock neurons was performed using the Bioconductor package edgeR (33 (link)). Raw data were submitted to the Gene Expression Omnibus (GEO) repository and are available using the accession number GSE202407.