Hiseq4000 sequencing instrument

The DNA of individuals and their parents was extracted from saliva samples, using the Oragene DNA Kit (DNA Genotek Inc., Ottawa, ON, Canada), or from blood samples using the Chemagic Magnetic Separation Module I (Cheagen, Baesweiler, Germany). In the first step, exome sequencing was performed for 14 CE case-parent trios and 1 affected CBE and E sib-pair at the Next-Generation Sequencing Laboratory of the Cologne Center for Genomics (CCG), using the Agilent SureSelect Human All Exon V6 for 12 families, the NimbleGen SeqCap EZ Human Exome Library v 2.0 for 2 families, and the Agilent SureSelect All Exon V7 for 1 family. After validation (2200 TapeStation; Agilent Technologies, Santa Clara, CA, USA) and quantification (Qubit System; Invitrogen, Waltham, MA, USA), pools of libraries were generated and subsequently sequenced on the Illumina HiSeq 4000 sequencing instrument, using a paired-end 2 × 100 bp protocol. The mean coverage of the presented exome data was 70,933 reads. In total, 92.31% of the targeted bases were covered by at least 20×. Since we filtered the reads with a minimum coverage of 10×, all the prioritized variants were validated using Sanger sequencing. Data analysis and filtering of the mapped target sequences were accomplished using the “Varbank” exome and genome analysis pipeline, version 2.0 (https://varbank.ccg.uni-koeln.de, accessed on 1 May 2000).

Total RNA was isolated from four human colon tissue samples using an RNeasy Blood and Tissue kit (Qiagen, Carlsbad, CA, USA). To construct the sequencing library for HiSeq 4000, we followed the TruSeq Stranded mRNA Sample Preparation Guide, Part #15031047 Rev. E. Approximately 2 μg of total RNA was used for library construction with the Illumina TruSeq Stranded mRNA Library Prep Kit (San Diego, CA, USA). Next, paired-end sequencing was performed using the Illumina HiSeq4000 sequencing instrument, according to the manufacturer’s instructions, yielding 101-bp paired-end reads. To construct the library for the MGISEQ-2000, approximately 1 μg of total RNA was used for library construction using the MGIEasy RNA Directional Library Prep Kit (MGI). Next, paired-end sequencing was performed using the MGISEQ-2000 sequencing instrument, according to the manufacturer’s instructions, yielding 100-bp paired-end reads. The RNA-seq data of HiSeq 4000 were generated in 2013, while the MGISEQ-2000 data were generated in 2019. Thus, although we used RNA from the same samples, the sequencing was not performed at the same time.

For the assessment of gene expression levels, the mice were sacrificed at 1, 6, 12, 24, and 48 h after BBBD (n = 2 per time point). The sham control mice received MB and contras agents but without FUS sonication (n = 2). The tissue was snap-frozen in liquid nitrogen and stored at− 80 °C until RNA isolation. According to the manufacturer’s instructions, tissues (100–120 mg) were homogenized and isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany). The total RNA concentration and quality were determined using a NanoDrop™ 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). Subsequently, total RNA was used for sequencing or real-time qPCR analyses. cDNA libraries were prepared with 1 μg of total RNA using the Illumina TruSeq RNA Sample Prep Kit v2 (Illumina Inc., San Diego, CA, USA). Next, paired-end sequencing was performed using the Illumina HiSeq^™4000 sequencing instrument, according to the manufacturer’s instructions, yielding 100-bp paired-end reads.