D1000 dna screentape

NEB Next Ultra RNA Library prep kit (NEB, Ipswich, MA, USA, #E7530L) was used to prepare the libraries for RNA sequencing. First, mRNA was selected using Poly-A selection kit (Lexogen, Vienna, Austria, # K39.100) from Lexogen. Following purification, the mRNA was fragmented using divalent cations under elevated temperatures. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase. Second-strand cDNA synthesis was performed using DNA Polymerase I and RNase H enzyme. The cDNA fragments were then subjected to a series of enzymatic steps that repair the ends, tailing the 3′ end with a single ‘A’ base, followed by the ligation of the adapters. The adapter-ligated products were then purified and enriched using the following thermal conditions: initial denaturation 98 °C for 30 s; 12 cycles of −98 °C for 10 s and 65 °C for 75 s; final extension of 65 °C for 5 min. PCR products are then purified and checked for fragment size distribution on TapeStation using D1000 DNA ScreenTapes (Agilent Technologies, Santa Clara, CA, USA, Cat# 5067-5582).

NEB Next Multiplex Small RNA Library Prep Kit was utilized to prepare the libraries. First, 200 ng of total RNA was denatured under elevated temperatures. The denatured RNA was then ligated to a 3ʹ SR adapter, followed by SR-RT primer annealing to convert the free single-stranded adapter to the double-stranded DNA molecule. Reverse transcriptase was used to copy the 3ʹ SR adapter-ligated molecules into first-strand cDNA. Following that, the adapter-ligated products were purified and enriched using the thermal conditions provided here: initial denaturation at 94 °C for 30 s; 15 cycles at 94 °C for 15 s, 62 °C for 30 s, 70 °C for 15 s; final extension at 70 °C for 5 min. After that, PCR products were purified, and fragment size distribution was examined on Tape Station using D1000 DNA Screen Tapes (Agilent, Cat# 5067–5582), accompanied by size selection on 4% E-gels. The Qubit High Sensitivity Assay (Invitrogen, Cat# Q32852) was used to quantify the libraries that had been produced. Before cluster amplification on the Illumina flow cell, the resulting libraries were merged and reduced to the final optimum loading percentage. The cluster flow cell is then put into the HiSeq 2500 instrument to generate 25 M 50 bp single-end reads.