Tiangen blood dna kit

After informed consent was obtained, whole blood was collected for whole exome Sequencing (WES). Genomic DNA was extracted from blood with the TIANGEN Blood DNA Kit (DP304, TIANGEN, China). Whole Exome Capture AIExomeV2 kit (iGeneTech Co., Beijing, China) was used for WES. The whole exon region was enriched using a liquid phase probe method and sequenced on the Illumina Nova sequencing platform (Illumina, Inc., California, USA) according to the manufacturer’s standard operating protocol. A depth of 100× was ensured in sequencing and the coverage of targeted exons was no less than 99%. Sequence data were uploaded to NCBI Sequence Read Archive with accession number SRR19049154.

A 2-ml whole fresh blood sample was collected from each patient in an EDTA tube for genotyping. DNA was extracted from peripheral blood samples using the Tiangen Blood DNA Kit (Tiangen Inc., Beijing, China). SLC22A1 rs622342 and ATM rs11212617 variants were analyzed by the dideoxy chain-termination method. The amplification primers used for polymerase chain reaction (PCR) were as follows: F 5′-CACCACATGAGTTAACAGCAGATT-3′ and R 5′-GCTCAAGCAAGCCTCCTACC-3′ for SLC22A1 rs622342; F 5′-CTCAATTAAAACCAGAGAAGGCAG-3′ and R 5′-AATTTTTTGCGTGGAGTCAGAGTC-3′ for ATM rs11212617. A 25 μl PCR mixture reaction system included 1 μl upstream primer (3.2 pmol/μL), 1 μl downstream primer (3.2 pmol/μL), 0.25 μl 2 × Taq DNA polymerase, 0.5 μl dNTP mixture, 1 μl DNA, and ddH₂O 21.25 μl. PCR reaction conditions included initial predenaturation at 95°C for 5 min, followed by 40 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 58°C, extension for 1 min at 72°C, with a final extension at 72°C for 5 min. The PCR product was electrophoresed by an ABI3730XL (Applied Biosystems, Inc., Carlsbad, California, America) sequencer after purified.

Genomic DNA was extracted from peripheral blood of all family members with the TIANGEN Blood DNA Kit (DP304, TIANGEN, China). Targeted next generation sequencing (NGS) was performed for the proband using a custom designed panel (PS400) containing 376 known genes associated with inherited retinal diseases (Zhu et al., 2021 (link)). The Nextseq 500 (Illumina, San Diego, CA, USA) platform was used for paired-end sequencing with read lengths of 150 bp and average sequencing depth of almost 300 X. Raw reads were mapped to the human genome reference (UCSC hg19) using three commercial software including XYGeneRanger 2.0 (Xunyin, Shanghai, China), TGex (LifeMap Sciences, Alameda, CA, USA) and Efficient Genosome Intepration System, EGIS (SierraVast Bio-Medical Technology Co., Ltd, Shanghai, China). Variant-filtering was based on public and in-house SNP databases, including 1000Genome project, HGMD, ExAC and ClinVar, as well as our internal database. The non-synonymous and splicing variants with MAF <2% were kept for further analysis. Sanger sequencing and co-segregation analysis were performed for the verification of suspicious disease-relevant gene variants in the available family members. Primer sequences and PCR conditions were shown in Supplementary Tables 1, 2, respectively.

Peripheral blood of all participants from the two adRP families was collected in EDTA anticoagulant tubes, and then, genomic DNA was isolated by using the TIANGEN Blood DNA Kit (TIANGEN, Beijing, China) according to the manufacturer's protocol. The RNA was extracted using TRIzol (TransGen, Beijing, China), then the total RNA (100 ng) of each sample was reverse‐transcribed to cDNA using the TransScript Reverse Transcription System (TransGen). DNA and cDNA integrity was detected using 1% of agarose gel electrophoresis.

Genomic DNA was isolated from peripheral blood of all subjects with the TIANGEN Blood DNA Kit (DP304, TIANGEN, Beijing, China). Nanodrop 2000 was used to detect the DNA concentrations. Concentrations above 25 μg/μl and OD260/280 between 1.8 and 2.0 were used for the next steps. Sequencing and alignment were performed on the Nextseq 500 (Illumina, San Diego, CA, USA) platform. Filter the original read to delete low quality reads (Q30 ≥ 80% was defined as qualified). Clean data were mapped using BWA (Burrows-Wheeler Alignment tool). The mtDNA was sequenced with a depth greater than X5000. The Sanger sequencing was conducted to verify disease-relevant mutations.