Agilent hs d1000 tape

Released ligated probe products were harvested from the slides and further processed to generate cDNA libraries for sequencing. To determine the sample index PCR cycle number, a qPCR was performed using Kappa Sybr master mix and primers (Roche, cat# 07959389001). The cDNA was then indexed and amplified. The indexed libraries were then cleaned, and size selected using SPRIselect (Beckman Coulter, cat# B23318). The average size of the indexed cDNA libraries was determined with an Agilent HS D1000 tape (Agilent, cat# 5067-5584) using Agilent tapestation (Agilent, cat# G2991BA). The molarity and concentrations of each library were measured using a qPCR, and libraries were pooled and diluted to 4 nM. Paired-end sequencing was performed on the Illumina NextSeq500 platform at 1.8pM loading concentration, with 29 bases from read 1 and 51 bases from read 2. A total of 80-100 million read pairs per sample were generated.

Nucleic acid enrichment was performed with the complimentary primer (JS15) without a tetradecamer at the 3′-end in a 50 μL reaction mixture consisting of 10 μL cDNA, 5 μL of 10 μM JS15 primer, 25 μL of TaqMan environmental master mix (version 2.0) PCR buffer (Life Technologies, Grand Island, NY) and 10 μL of nuclease free water. After an initial denaturation step at 95 °C for 10 min, PCR was performed for 30 s at 95 °C, 45 s at 60 °C, and 45 s at 72 °C for 45 cycles followed by a final extension step at 72 °C for 10 min. The amplification products were purified with AMPure SPRIselect beads (Beckman Coulter, Indianapolis, IN), and a 1:0.8 ratio was selected to obtain an average DNA fragment size of 392 bp as confirmed by the Agilent 4200 Bioanalyzer TapeStation using the Agilent HS D1000 tape. DNA concentration was determined using the Qubit dsDNA HS kit with the Qubit 3.0 Fluorimeter (ThermoFisher Scientific, Waltham, MA).

Nucleic acid enrichment was performed with the complimentary primer (JS15) without a tetradecamer at the 3′-end in a 50 μL reaction mixture consisting of 10 μL cDNA, 5 μL of 10 μM JS15 primer, 25 μL of TaqMan environmental master mix (version 2.0) PCR buffer (Life Technologies, Grand Island, NY) and 10 μL of nuclease free water. After an initial denaturation step at 95 °C for 10 min, PCR was performed for 30 s at 95 °C, 45 s at 60 °C, and 45 s at 72 °C for 45 cycles followed by a final extension step at 72 °C for 10 min. The amplification products were purified with AMPure SPRIselect beads (Beckman Coulter, Indianapolis, IN), and a 1:0.8 ratio was selected to obtain an average DNA fragment size of 392 bp as confirmed by the Agilent 4200 Bioanalyzer TapeStation using the Agilent HS D1000 tape. DNA concentration was determined using the Qubit dsDNA HS kit with the Qubit 3.0 Fluorimeter (ThermoFisher Scientific, Waltham, MA).