For validation of splicing events from different sources, RNA was extracted from MEFs, embryonic E18.5 head or adult spinal cord. RNA quality was determined by RNA Integrity Number (RIN) measured using RNA ScreenTape and reagents (Agilent) on the 2200 Tapestation System (Agilent). RT–PCR protocol was conducted following manufacturer's instructions (Thermo, QuantaBio, Applied Biosystems). PCR for splicing events was conducted using 2X PCR Master Mix (Thermo) with specific primers spanning the differentially expressed exon (RRM2mut cryptic n = 4, LCDmut skiptic n = 4, Q331K splicing/skiptic n = 3). Products were electrophoresed on agarose gels with ethidium bromide (Sigma) or using D1000 ScreenTape and reagents (Agilent) on the 2200 Tapestation System (Agilent). Results were analysed using PSI, calculated dividing the intensity of the exon inclusion band by the sum of both exon inclusion and exon exclusion bands. When analysing SEs in human fibroblasts, as a combination of three primers was used, we did not calculate PSI, but calculated the ratio between SE band and the exon inclusion band and expressed results after normalizing the mean of healthy controls to 100%. Primer sequences are available upon request.
2200 tapestation system
Manufactured by Agilent Technologies
Sourced in United States, Germany
The 2200 TapeStation system is a lab equipment product from Agilent Technologies. It is designed to provide automated sample quality assessment and quantification for nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (15 mentions)
Hiseq 2500, by Illumina (14 mentions)
Rneasy mini kit, by Qiagen (13 mentions)
D1000 screentape, by Agilent Technologies (12 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (12 mentions)
Kapa library quantification kit, by Roche (11 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (9 mentions)
Hiseq 4000, by Illumina (9 mentions)
Nanodrop nd 2000, by Thermo Fisher Scientific (8 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (7 mentions)
Agilent rna screentape, by Agilent Technologies (6 mentions)
Rneasy kit, by Qiagen (6 mentions)
Novaseq 6000, by Illumina (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Qiazol lysis reagent, by Qiagen (5 mentions)
Ampure xp bead, by Beckman Coulter (5 mentions)
Nextseq 500, by Illumina (5 mentions)
Genechip fluidics station 450, by Thermo Fisher Scientific (4 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (4 mentions)
Allprep dna rna protein mini kit, by Qiagen (4 mentions)
207 protocols using 2200 tapestation system
1
Validating Splicing Event Profiles
2
RNA-Seq Library Preparation and Sequencing
3
Small RNA-Seq Library Preparation
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To test the quality and assess the quantity of total RNA extracted, we used the RNA ScreenTape assay on a 2200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA). For small RNA-Seq, 1 μg of total RNA per sample was used for library preparation using TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, CA, USA). Size-distribution was measured with the DNA ScreenTape assay on a 2200 TapeStation system (Agilent Technologies, Santa Clara, CA, USA). A total library pool of 4 nM was sequenced using a MiSeq Reagent Kit v3 150 cycle on a MiSeq System (Illumina, San Diego, CA, USA).
4
RNA-Seq Library Preparation and Sequencing
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RNA was purified using the RNeasy Mini Kit (Qiagen). RNA quality was evaluated using a 2200 TapeStation system (Agilent), and samples with RNA integrity score score ≥ 7 were selected for library preparation. RNA-Seq libraries were prepared and amplified according to KAPA Stranded mRNA-Seq Kit-Illumina Platforms (KAPA Biosystems), using Oligo-dT magnetic beads to enrich for mRNA. Sample library fragment sizes were confirmed using a 2200 TapeStation system (Agilent) and quantified by qPCR using the Library Quantification kit (KAPA Biosystems). Library adaptors 1–12 (KAPA Biosystems) were used to barcode each sample, and libraries were sequenced on a HiSeq 2000 sequencing system (Illumina) in a 100-bp, paired-end run.
5
Whole Exome Sequencing Library Preparation
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To prepare libraries for Whole Exome Sequencing, 100ng-1µg of genomic DNA was sheared with the Covaris S220 (Covaris, Woburn, MA, USA), followed by end-repair, 3’ end Adenylation and ligation with paired-end adaptors. Post ligation, 15µl of the purified libraries were PCR amplified, all the above steps were performed using the Agilent SureSelectXT kit and every step was followed by DNA purification on a magnetic stand using AMPure XP Reagent beads (Beckman Coulter Genomics, Danvers, MA, USA). Afterward, size (approx 225-275 bp) and quantity (>800ng) were verified employing the Agilent Tapestation 2200 system followed by hybridization and probe capture using Exome SureSelect Human All Exon V6+UTR probes (Agilent). Dynabeads MyOne Streptavidin T1 magnetic beads (Thermofisher). Finally, Captured Libraries were amplified with 12 cycles of PCR using indexing primers containing 8-bp indices, followed by an amplification using AMPure XP beads (Beckman Coulter). Final libraries were checked for quality (each fragment size approx. 300-400 bp) and quantity using Agilent Tapestation 2200 system.
6
RNA Isolation and Sequencing Library Preparation
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Total RNA was isolated using the miRNeasy Micro Kit (QIAGEN GmbH, Germany) according to the manufacturer’s guidelines. RNA concentration and quality was assed using the Tapestation 2200 system and high sensitivity reagents (Agilent Technologies, USA). Sequencing libraries of FACS-enriched populations were prepared following the QIAseq FX Single Cell RNA Library Kit protocol (QIAGEN GmbH, Germany) and libraries from cultured PBMC were generated according to the Smart-seq2 protocol (Picelli et al., 2014 (link)). For QIAseq FX Single Cell RNA Libraries, concentration of cDNA was assessed by KAPA Library Quantification Kits (Kapa Biosystems, USA) before sequencing single read 75bp on a HiSeq1500 instrument using TruSeq SBS v3-HS chemistry. For Smart-seq2 libraries, concentrations were determined using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA) and average library size was determined using an High Sensitivity D5000 assay on a Tapestation 2200 system (Agilent Technologies, USA) before pooling and sequencing single read 75bp on a NextSeq500 system using High Output v2 chemistry.
7
Micrococcal Nuclease Digestion and Sequencing
8
Illumina Sequencing of Bacterial Genomes
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Genomic DNA libraries were prepared for all Australian isolates using an Illumina Nextera™ DNA Flex Library Prep kit with CD indexes according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). The size distribution of libraries was determined using the HSD1000 ScreenTape device on the 2200 TapeStation system (Agilent technologies). Concentration was determined using the Qubit Fluorometer2.0 (Invitrogen) and KAPPA Library Quantification kit (KapaBiosystem) according to the manufacturer’s instructions. Pooled libraries were sequenced (2 × 250 bp paired end reads) using a Miseq v3 reagent kit on an Illumina Miseq® platform (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions. Illumina reads were quality trimmed using Trimmomatic [70 ] with the following parameters: > 15 quality score, sliding window of 4 and minimum length of 200 bp.
ANI between all possible pairs of 50 representative assembled genomes was calculated using pyani [71 ] and visualised using Pheatmap v1.2.12 in R. Quast version 5 was used to evaluate the assemblies and provide genome metrics including contig number, GC content and N50 values [72 (link)].
ANI between all possible pairs of 50 representative assembled genomes was calculated using pyani [71 ] and visualised using Pheatmap v1.2.12 in R. Quast version 5 was used to evaluate the assemblies and provide genome metrics including contig number, GC content and N50 values [72 (link)].
9
Optimized gDNA Extraction for Cells
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We extracted gDNA from the cultured cells and the clinical specimens using the MagAttract HMW DNA Kit (Qiagen) or smart DNA prep (a) (Analytik Jena) according to the manufacturer's instructions. Extracted gDNA was quantified using Genomic DNA ScreenTape assay and a 2200 TapeStation system (Agilent Technologies). We only used gDNA for which the DNA integrity number (DIN) was ∼9 except for the tumor tissue of Case 8, for which the DIN was 6.6.
10
Nextera XT Library Prep for RNA-seq
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Library preparation of suitable cDNAs were performed using Nextera® XT Library Prep Kit (Illumina, CA) with Nextera® XT Index Kit V2 Set A (Illumina, CA). Samples were normalized to 40 pg/μl for a total 200 pg input of amplified cDNA. The protocol was performed as described by the manufacturer. Libraries were purified with 0.6 x bead ratio using Agencourt AMPure beads XP (Beckman Coulter, IN) and eluted in 52.5 μl of elution buffer. Quality parameters as size (440 bp average) and concentration (1.03 ng/μl average) were measured using High Sensitivity D1000 ScreenTape and reagents run on 2200 TapeStation System (Agilent, CA) and Qubit™ dsDNA HS Assay Kit on a Qubit 3 Fluorometer (Thermo Fisher Scientific, MA), respectively. Good quality libraries were normalized to 1 nM. Thirteen samples were pooled to further perform 101 cycles of single read sequencing using a NextSeq® 500/550 High Output Kit v2 (150 cycles) and NextSeq® 550 sequencer (Illumina, CA). Sixty-two libraries with more than 20 million reads each were considered for analysis.
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