3 ng/ul of pooled cDNA library was used as sequencing input for each Smart-3SEQ libraries. Smart-3SEQ cDNA libraries were prepared with a 40% spike-in of phiX and sequenced on Illumina NovaSeq 6000 SP Flowcell instrument (approximately 400M PE Reads per lane) configured for 100nt read 1 and 8nt for the P7 index read. TruSeq mRNA-seq libraries were sequenced on two lanes of an Illumina HiSeq 4000 in 75PE configuration. 40% PhiX was added out of an abundance of caution during method implementation. This is due to our use of the two-color Illumina Novaseq 6000 system, which may generate errors when sequencing SMART-3SEQ libraries due to a trinucleotide G repeat in the second strand template-switching reverse transcription primer. We did not systematically investigate lower amounts of PhiX, which would further decrease sequencing costs.
Novaseq 6000 sp flowcell instrument
Manufactured by Illumina
The NovaSeq 6000 SP Flowcell instrument is a high-throughput sequencing platform produced by Illumina. The core function of this equipment is to enable the sequencing of DNA samples using a flow cell-based approach.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using novaseq 6000 sp flowcell instrument
1
Smart-3SEQ Library Preparation and Sequencing
2
Smart-3SEQ RNA-Seq Library Preparation
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Extracted RNA and cell lysate (above) was used for preparing sequencing libraries according to the Smart-3SEQ protocol (10) . Illumina TruSeq libraries were prepared at the University of Iowa Genomics Facility using standard Illumina TruSeq stranded mRNA protocols. Concentration and fragment size of prepared library was measured using Qubit HS and bioanalyzer. Fragment size for Smart-3SEQ library was ~500bp which falls within the recommended 200-600bp.
Sequencing: Smart-3SEQ cDNA libraries were prepared with a 40% spike-in of phiX and sequenced on Illumina NovaSeq 6000 SP Flowcell instrument (approximately 400M PF Reads per lane) configured for 100nt read 1 and 8nt for the P7 index read. TruSeq mRNA-seq libraries were sequenced on a single lane of an Illumina HiSeq 4000 in 75PE configuration.
Sequencing: Smart-3SEQ cDNA libraries were prepared with a 40% spike-in of phiX and sequenced on Illumina NovaSeq 6000 SP Flowcell instrument (approximately 400M PF Reads per lane) configured for 100nt read 1 and 8nt for the P7 index read. TruSeq mRNA-seq libraries were sequenced on a single lane of an Illumina HiSeq 4000 in 75PE configuration.
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