Dna 1000 nano lab chip kit

Total RNA was isolated from all samples using the Qiagen RNeasy Plus Universal kit (Qiagen, UK) in accordance with the manufacturer’s instructions. Following isolation, RNA samples were quantified on the Nanodrop spectrometer, and RNA quality was assessed on the Agilent Bioanalyzer using the RNA 6000 bioanalyzer kit. Only samples with RNA integrity number (RIN) values greater than 8 were used for subsequent RNA-sequencing. cDNA libraries were prepared from 1 µg of total RNA for each sample using the Illumina Truseq stranded mRNA kit (Illumina, San Diego, CA, USA). Briefly, using 1 µg of total RNA as starting material for each sample, mRNA was purified and subsequently fragmented. SuperScript II Reverse Transcriptase (Applied Biosystems Ltd.) was used for first strand cDNA synthesis from the purified mRNA, with the second strand of cDNA synthesized using components of the Illumina Truseq kit. Following ligation of sequencing adapters, cDNA libraries were then enriched through PCR. Final cDNA libraries were then validated using the DNA 1000 Nano Lab Chip kit on the Agilent Bioanalyzer 2100, ensuring that library fragment size was ~ 260 bp and library concentration was > 30 ng/µl. Sequencing of cDNA libraries was then undertaken on an Illumina Novaseq platform employing 150 bp paired-end sequencing.

cDNA libraries were prepared from high quality RNA (3 μg per sample) using an Illumina TruSeq RNA sample prep kit following the manufacturer’s instruction (Illumina, San Diego, CA, USA). Briefly, mRNA was isolated from total RNA and strands subsequently fragmented. First strand cDNA was synthesised using SuperScript^® II Reverse Transcriptase (Applied Biosystems Ltd., Life Technologies, Warrington, UK), second strand synthesis was subsequently performed using components supplied in the Illumina TruSeq RNA sample prep kit. Indexing adaptors were ligated to the cDNA which was then enriched through PCR. Final individual cDNA libraries were validated on the Agilent Bioanaylser 2100 using the DNA 1000 Nano Lab Chip kit, ensuring that library fragment size was ~260 bp and library concentration was >30 ng/μl. After quality control procedures, individual RNA-seq libraries were pooled based on their respective sample-specific-6bp adaptors and sequenced at 100 bp/sequence on an Illumina HiSeq 2000 generating single-end reads.

Twenty cDNA libraries were prepared from high quality RNA using an Illumina TruSeq RNA Sample Preparation kit v2 following the manufacturer’s instructions (Illumina, San Diego, CA, USA). For each sample, 1 μg of total RNA was used for cDNA preparation. All libraries were validated on the Agilent Bioanalyzer 2100 using the DNA 1000 Nano Lab Chip kit (Agilent Technologies Ireland Ltd., Dublin, Ireland). Individual RNAseq libraries were pooled (10 libraries per lane) based on their respective sample-specific-6 bp adaptors and sequenced at 100 bp/sequence single-end reads using an Illumina HiSeq 2500 sequencer.

cDNA libraries were prepared from high quality RNA following the manufacturer’s instructions using the Illumina TruSeq mRNA sample prep kit (Illumina, San Diego, CA, USA). For each sample, 1 μg of RNA was used for cDNA library preparation. Resultant cDNA libraries were validated on the Agilent Bioanalyser 2100 using the DNA 1000 Nano Lab Chip kit. cDNA concentration was assessed using Nanordrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and samples with > 25 ng/μl were deemed suitable for further analysis. Following quality control procedures, individual RNAseq libraries were pooled based on their respective sample-specific 6 bp adaptors and sequenced at 100 bp/sequence single-end read using and Illumina HiSeq 2500 sequencer. Approximately 22.4 million sequences per sample were generated.

cDNA libraries were prepared from high quality RNA following the manufacturer’s instructions using the Illumina TruSeq stranded mRNA sample prep kit (Illumina, San Diego, CA, USA). For each sample, 1 μg of RNA was used for cDNA library preparation. Briefly, mRNA was purified from total RNA and subsequently fragmented. First strand cDNA was synthesised using Superscript II Reverse Transcriptase (Applied Biosystems Ltd., Life Technologies) followed by second strand synthesis using components of the Illumina TruSeq RNA sample prep kit. Adaptors were ligated to the cDNA which was subsequently enriched by 15 cycles of PCR. Libraries were validated on the Agilent Bioanalyser 2100 using the DNA 1000 Nano Lab Chip kit. cDNA concentration was assessed using Nanordrop spectrophotometer ND-1000 (Nanodrop Technologies, Wilmington, DE, USA) and samples with > 25 ng/μl were deemed suitable for further analysis. Following quality control procedures, individual RNA-seq libraries were pooled based on their respective sample-specific 6 bp adaptors and sequenced at 50 bp/sequence single-end read using an Illumina HiSeq 2500 sequencer. Approximately 22 million sequences per sample were generated.

cDNA libraries were prepared from high quality RNA samples using the Illumina TruSeq RNA sample prep kit following the manufacturer’s instructions (Illumina, San Diego, CA, USA). For each sample, 3 µg of RNA was used for cDNA preparation. Individual cDNA libraries prepared from mRNA were validated through the DNA 1000 Nano Lab Chip kit on the Agilent Bioanalyser 2100 ensuring that library fragment size was ~ 260 bp and library concentration was > 30 ng/µl. Individual RNAseq libraries were then sequenced on an Illumina HiSeq 2000 sequencer, resulting in 100 bp single end sequencing reads.