RNA samples containing a minimum of 1 ng RNA and a RIN > 4 were used for further RNAseq analysis. First, RNA was amplified using the Ovation® SoLo RNA-seq library preparation kit (NuGEN) used for low input or single-cell sequencing according to manufacturer’s instructions, including a ribosomal RNA depletion step (Insert Dependent Adaptor Cleavage). Libraries were then monitored for concentration and fragment sizes using a Fragment Analyzer (kit Standard Sensitivity NGS) and by qPCR (ROCHE Light Cycler 480) prior sequencing on Illumina HiSeq2000 platform (single end 50 bp length).
Lightcycler 480
Manufactured by Illumina
Sourced in United States
The LightCycler 480 is a real-time PCR system designed for high-throughput quantitative and qualitative nucleic acid analysis. The system utilizes a 96-well or 384-well microplate format and provides precise temperature control and monitoring for reliable and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (2 mentions)
Nextseq 550, by Illumina (2 mentions)
Nebnext ultra 2 directional rna library kit, by New England Biolabs (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Smart seq v4 ultra low input rna kit for sequencing, by Takara Bio (2 mentions)
Kapa library quantification kit, by Illumina (2 mentions)
Fragment analyzer, by Agilent Technologies (2 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Ovation solo rna seq library preparation kit, by Tecan (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Ht dna hisens reagent kit, by PerkinElmer (1 mentions)
Le220 focused ultrasonicator, by Covaris (1 mentions)
Truseq pcr free library preparation, by Illumina (1 mentions)
Nebnext ultra 2 fs dna library kit, by Illumina (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Purelink genomic dna mini kit, by Thermo Fisher Scientific (1 mentions)
Hiseq x10, by Illumina (1 mentions)
7 protocols using lightcycler 480
1
Low-input RNA-seq Library Preparation
2
RNA Extraction and RNA-Seq Workflow
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Pancreata were placed in RNAlater (Invitrogen) overnight at 4°C. Samples were homogenized in TRIzol (Thermo Fisher Scientific), and 100 μL chloroform was added for each milliliter of TRIzol. The top aqueous layer was collected, and an equal volume of 70% ethanol was added. RNA was isolated using RNeasy Mini Kit (QIAGEN). For organoids, cells were first dissociated, and RNA was extracted as above. RNA-Seq was conducted using Illumina’s NextSeq550 and NovaSeq 6000 at the UAB Genomics Core. RNA quality was assessed using the Agilent 2100 Bioanalyzer, and RNA with an RNA integrity number ≥ 7.0 was used for library preparation. RNA was converted to a sequencing-ready library using the NEBNext Ultra II Directional RNA library kit (New England Biolabs). The cDNA libraries were quantitated using quantitative PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation.
3
RNA Extraction and RNA-Seq Workflow
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Pancreata were placed in RNAlater (Invitrogen) overnight at 4°C. Samples were homogenized in TRIzol (Thermo Fisher Scientific), and 100 μL chloroform was added for each milliliter of TRIzol. The top aqueous layer was collected, and an equal volume of 70% ethanol was added. RNA was isolated using RNeasy Mini Kit (QIAGEN). For organoids, cells were first dissociated, and RNA was extracted as above. RNA-Seq was conducted using Illumina’s NextSeq550 and NovaSeq 6000 at the UAB Genomics Core. RNA quality was assessed using the Agilent 2100 Bioanalyzer, and RNA with an RNA integrity number ≥ 7.0 was used for library preparation. RNA was converted to a sequencing-ready library using the NEBNext Ultra II Directional RNA library kit (New England Biolabs). The cDNA libraries were quantitated using quantitative PCR in a Roche LightCycler 480 with the Kapa Biosystems kit for Illumina library quantitation.
4
RNA-seq Analysis of PSC Lineages
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Samples were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech), according to manufacturer’s protocols and using 10 ng of input. Final libraries were evaluated for size using a Fragment Analyzer (Advanced Analytical Technologies) and quantified using both Qubit (Thermo Fisher) and qPCR (Roche LightCycler 480, KAPA Illumina Library Quantification Kit) before sequencing. RNA-seq 75 bp paired-end reads from Illumina were checked with FastQC and fastq_screen. Reads were mapped to human genome hg19 using TopHat v 2.0.13 with Ensembl annotation (GRCh37.75) in gtf format. FeatureCounts was used to obtain gene counts with default option. The gene counts were normalized with DESeq, and PCA plot was created with plotPCA function. Bar charts represent the average normalized raw reads for different PSC lines and primary samples. Gene ontology differently selected top features for FMG, pMGLs and differentiated NPCs, ran on the online Gene Ontology Consortium tool (geneontology.org). Hierarchical clustering was performed on quantile normalized FPKMs from the different GEO datasets. For HMDM and iPSDM, we used GSE55536. For the comparison to adult human microglia, and other brain cells, we used GSE73721. Biological replicates of pMGLs, FMGs and differentiated NPCs were used for analyses. The values for pMGL3 were the average of two technical replicates.
5
RNA-seq Analysis of PSC Lineages
6
High-Coverage Sheep Genome Sequencing
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High quality genomic data were extracted from ear notch biopsies for 130 sheep using a Nucleospin Tissue Kit and quality checked using an Agilent Tapestation 2200. For library preparation, 1 µg of gDNA was sheared to fragments of 450 bp mean size using a Covaris LE220 focused-ultrasonicator. DNA fragments were blunt ended, A-tailed, size selected and adapters ligated onto fragment ends according to Illumina TruSeq PCR-free library preparation kit protocol. Insert size on the libraries was evaluated using a PerkinElmer LapChip GX Touch with an HT DNA 1k/12K/HI SENS LabChip and HT DNA HI SENS Reagent Kit. Final library concentration was calculated by qPCR using a Roche LightCycler 480 and a Kapa Illumina Library Quantification kit and Standards. Then libraries were normalized to a loading concentration of 150 nM. All the library processing steps were carried out on Hamilton MicroLab STAR liquid handling robots coupled to BaseSpace Clarity LIMS X Edition. Libraries for all samples were loaded into a HiSeq X Flow cell v2.5, and clustered using an Illumina cBot2 Cluster Generation System. All libraries were sequenced on the HiSeqX to a mean coverage of 54X with 150 bp paired-end reads.
7
Genomic DNA Extraction and Sequencing
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Genomic DNA was extracted from single-colony cultures using PureLink™ Genomic DNA Mini Kit (Invitrogen, USA). The DNA integrity was checked by electrophoresis in a 1% (w/v) agarose gel, the DNA purity was estimated using NanoDrop® spectrophotometer (Nanodrop Technologies Inc., USA), and the concentration was determined with a Qubit Fluorometer (Invitrogen, USA).
Subsequently, 1000 ng of DNA was shipped to the Wellcome Sanger Institute (Hinxton, UK) to sequence using Illumina HiSeq-X10 (San Diego, USA). DNA concentrations were confirmed using AccuBlue Broad Range assay (Biotium, Inc., Fremont, USA), followed by normalization and DNA library construction. DNA was sheared into 450–550 bp fragments using Covaris LC 220 ultrasonicator (Brighton, UK), followed by polymerase chain reaction (PCR)-based library preparation using Illumina adaptors and 384-indexed tags (NEBNext Ultra II FS DNA library kit). Afterward, size-selection, amplification, purification, and multiplexing were carried out, and libraries were pooled. Pool was quantified and normalized down to 4 nM using Biomek NXP workstation for (automated liquid handling; California, USA), Agilent Bioanalyzer 2100 (California, USA), and Roche Lightcycler 480) (Utah, USA), before denaturation and loading on the Illumina platform.
Subsequently, 1000 ng of DNA was shipped to the Wellcome Sanger Institute (Hinxton, UK) to sequence using Illumina HiSeq-X10 (San Diego, USA). DNA concentrations were confirmed using AccuBlue Broad Range assay (Biotium, Inc., Fremont, USA), followed by normalization and DNA library construction. DNA was sheared into 450–550 bp fragments using Covaris LC 220 ultrasonicator (Brighton, UK), followed by polymerase chain reaction (PCR)-based library preparation using Illumina adaptors and 384-indexed tags (NEBNext Ultra II FS DNA library kit). Afterward, size-selection, amplification, purification, and multiplexing were carried out, and libraries were pooled. Pool was quantified and normalized down to 4 nM using Biomek NXP workstation for (automated liquid handling; California, USA), Agilent Bioanalyzer 2100 (California, USA), and Roche Lightcycler 480) (Utah, USA), before denaturation and loading on the Illumina platform.
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