IFN-signaling gene (ISG) scores are a diagnostic biomarker of AGS. This cohort included the first encounter at which ISG scores were obtained concurrently with clinical evaluations (n = 36). ISG scores (z-scores) were calculated as available from each clinical visit where blood samples were obtained. These scores were derived from the mRNA measurement of 6 IFN-inducible genes (IFI27, IFI44L, IFIT1, ISG15, RSAD2, and SIGLEC1) and 4 housekeeping genes (ALAS1, HPRT1, TBP, and TUBB) as previously described [4 (link)]. Briefly, patient blood samples were collected in PAXgene blood RNA tubes (PreAnalytiX), and RNA was purified using PAXgene blood RNA kits (Qiagen). RNA quality was assessed by RNA TapeStation (Agilent) and the RIN number of all of the samples was above 6.0. The concentration of RNA was quantified by Qubit High Sensitivity RNA assay (Thermo), and 200 ng RNA was used for each sample in the nCounter™ Digital Analyzer.
Rna tapestation
Manufactured by Agilent Technologies
The RNA TapeStation is a lab equipment product from Agilent Technologies. It is designed to analyze the quality and quantity of RNA samples. The device uses microfluidic technology to assess the integrity of RNA samples in a rapid and automated manner.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Direct zol kit with dnase treatment, by Zymo Research (2 mentions)
Qubit rna high sensitivity assay, by Thermo Fisher Scientific (1 mentions)
Paxgene blood rna tube, by Preanalytix (1 mentions)
Paxgene blood rna kit, by Qiagen (1 mentions)
Qiashredder column, by Qiagen (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nanodrop, by Agilent Technologies (1 mentions)
Rlt buffer, by Qiagen (1 mentions)
Geneart, by Thermo Fisher Scientific (1 mentions)
Megaclear transcription clean up kit, by Thermo Fisher Scientific (1 mentions)
T7 mscript standard mrna production system kit, by CellScript (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Superscript 4, by Thermo Fisher Scientific (1 mentions)
Image lab, by Bio-Rad (1 mentions)
Mirneasy, by Qiagen (1 mentions)
High sensitivity, by Agilent Technologies (1 mentions)
Smrtbell express template prep kit 2, by Pacific Biosciences (1 mentions)
Sequel 2 system, by Pacific Biosciences (1 mentions)
10 protocols using rna tapestation
1
Diagnostic Biomarker for Aicardi-Goutières Syndrome
2
Macrophage Differentiation and RNA-Seq
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pMacpre were seeded at 1.5 × 106 cells/well in 6-well plates at 7, 8, and 9 weeks after setting up differentiation factories, and they were differentiated in macrophage media for 7 days. Cells were washed once with PBS, aspirated thoroughly, and lysed by addition of 350 μL Buffer RLT (QIAGEN) with 1% (v/v) 2-mercaptoethanol. Plates were stored at − 80 °C until all samples were collected, and then lysates were passed through QIAshredder columns (QIAGEN) and the total RNA extracted using a QIAGEN RNeasy Mini kit, according to the manufacturer’s protocol using on-column DNase treatment (QIAGEN). RNA samples were eluted in 30 μL of RNase-free water. The quantity and quality of RNA was measured by Nanodrop and RNA Tapestation (Agilent), with measured RNA integrity (RIN) values ≥ 8.4. Poly-A library preparation and sequencing (HiSeq 4000, 75 bp paired-end reads), and basic data processing, was conducted at the Oxford Genomics Centre, Wellcome Centre for Human Genetics.
3
Codon-optimized Construct Synthesis and mRNA Production
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Human codon-optimized constructs were synthesized (GeneArt, Thermo Fisher Scientific, Waltham, MA) and cloned into the mammalian expression vector pCI (Promega, Madison, WI). Per manufacturers’ instructions, the T7 mScript Standard mRNA Production System Kit (CELLSCRIPT, Madison, WI) was used to synthesize Cap 1 mRNA, followed by purification using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific, Waltham, MA). Prior to electroporation, RNA TapeStation (Agilent, Santa Clara, CA) analysis was performed to determine mRNA purity, integrity, and transcript size.
4
Poly-A RNA Extraction and Quality Control
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At 80% confluency in 10cm plates, cells were washed with PBS and harvested in 1mL of TRIzol reagent (Thermo Fisher) or Direct-zol kit with DNase treatment (Zymo Research). Total RNA was extracted following the manufacturer’s protocol. 20ug of total RNA was poly-A selected using a poly-A magnetic resin kit (NEB E7490L). RNA was then analyzed by high-sensitivity RNA Tapestation (Agilent #5067–5579) to confirm poly-A selection and RNA quality.
5
High-quality RNA-seq protocol for gene expression analysis
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Rigorous quality control checks were applied to assess the purity and integrity of RNA (Agilent BioAnalyzer; RNA TapeStation) RIN values ranged between 8.4 and 8.8 (see Supplementary Table 3 ). Illumina TruSeq Stranded mRNA kits were used to generate Poly(A) enriched bulk RNA-sequencing libraries. These libraries were loaded onto lanes of an Illumina NextSeq flowcell and sequenced using 75 bp paired-end (PE) runs (Source Bioscience). Each individual sample generated >35 million PE reads. Data were analysed as described in detail53 (link). The analysis was sufficiently powered (n = 5 per group) to reduce the false discovery rate and to enable systems-level analysis97 (link). Differentially expressed genes (DEGs) with P-adjusted (Padj) values of <0.05 are considered significant.
6
Quantitative Analysis of Xbp1 Splicing
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Total RNA was isolated from PWS and control INS-1 lines grown as above (7.5 mM glucose) by Trizol harvest and miRNeasy (Qiagen) column purification. RNA quality was assessed by RNA TapeStation (Agilent) and quantified by broad range Qubit (Thermo Fisher) fluorometric analysis. First strand cDNA synthesis from 1 μg RNA was carried out using random hexamer primed RT by using Super Script IV (Thermo Fisher). Primers for gene specific RT-PCR amplification are in S8 Table
7
Ribo-STAMP and APOBEC1 cell lines
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Plasmid construction, cell culture conditions and maintenance, and generation of doxycycline (dox)-inducible HEK293XT Ribo-STAMP (RPS2-APOBEC1) and APOBEC1-only stable cell lines were completed in accordance with methods outlined by Brannan et al. (2021) (link). For stable cell Ribo-STAMP and APOBEC1-only protein expression, cells were induced with 1ug/mL dox for 72h. Total RNA was isolated from technical triplicate samples of HEK293XT cells expressing Ribo-STAMP and APOBEC1-only constructs using TRIzol extraction and column purification using the Direct-zol Miniprep kit (Zymo Research). Poly(A) selection was completed using the Poly(A) mRNA Magnetic Isolation Module (NEB E7490L) and RNA quality was assessed using high-sensitivity RNA Tapestation (Agilent, . Long-read RNA-seq libraries were prepared using the PacBio Iso-Seq Express protocol (101-763-800) and PacBio SMRTbell Express Template Prep Kit 2.0 (100-938-900). Samples were barcoded using the PacBio Barcoded Overhang Adapter Kit (101-791-700) and then pooled in an equimolar fashion. Samples were sequenced on a SMRT cell 8M with a 30-hour movie time on the PacBio Sequel II system.
8
Poly-A RNA Extraction and Quality Control
9
Macrophage Phagocytosis of S. aureus
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Peritoneal macrophages were incubated with heat-treated S. aureus (wood strain, without protein A) for 1 h. The Promega Reliaprep cell miniprep system (TM370) was used to lyse cells and extract RNA. RNA quality was assessed via Agilent RNA TapeStation. The concentration of the RNA was quantified using a Qubit and the sample concentration was adjusted to 20 ng/μL in 10 μL. The RNA was then run on a NanoString nCounterTM (NanoString Technologies) in conjunction with a custom panel of phagocytosis genes.
10
Transcriptome Analysis of Sea Urchin Embryo Development
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Adult S. purpuratus were obtained from Pat Leahy (Caltech). Gametes were collected by intra-coelomic injection of 0.5 M KCl. Fertilization and embryo culture were performed according to the method of Adams et al. (2019) (link). Embryos were cultured at 15°C in 4-liter plastic beakers fitted with battery-powered stirrers. Embryos were harvested at 0, 6, 12, 18, 24, 30, 36, 42, and 48 hpf. To provide sufficient material for the analysis, two fertilizations were performed at different times on the same day using eggs from two different females and sperm from the same male. Culture 1 was used for seven time points, and culture 2 was used for two time points (18 hpf and 42 hpf). Cultures were not pooled at any time point. For the 0-hpf time point, embryos were collected immediately after fertilization. Total RNA (75–100 µg per sample) was isolated using the RNeasy Plus Mini Kit (Qiagen 74134), and the quality of the preparations was confirmed using an RNA TapeStation (Agilent). RNA integrity numbers (RINs) for all samples were 9.8–10.0. RNA samples were stored at −80°C.
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