Fragment analyzer hs ngs fragment kit

Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10 × Genomics, Pleasanton, CA) was used to prepare the sequencing libraries, and the protocol was performed according to the manufacturer’s instructions. Briefly, 10 × Chromium platform was used to encapsulate individual cells into droplets along with beads covered in cell-specific 10 × Barcodes, unique molecular identifiers (UMIs) and poly(dT) sequences. After reverse transcription, the cDNA libraries were amplified (13–14 cycles), fragmented and ligated to sequencing adaptors. SPRISelect magnetic beads were used for purification of the cDNA suspension and size selection of the fragments. Concentration and quality of the libraries was measured using Qubit dsDNA HS Assay Kit (Invitrogen) and Fragment Analyzer HS NGS Fragment Kit (#DNF-474, Agilent). The libraries were pooled and sequenced in paired-end mode using Illumina NovaSeq 6000 SP Reagent Kit, Read 1 containing a barcode and a UMI, and Read 2 covering the sequence of interest. Sequencing data comprised of approx. 100–200 million reads per sample (Supp. Tab. 4).

First-strand synthesis was generated by mixing 5 µL of RNA with 500 nM of reverse primer in a 20 µL reverse transcription reaction (Bio-Rad, 1708897) containing 4 µL of reaction supermix, 2 µL of GSP enhancer solution, and 1 µL of reverse transcriptase. The mixture was incubated at 42 °C for 30 or 60 min, followed by a deactivation of the reverse transcriptase at 85 °C for 5 min. To generate ample product for gel electrophoresis analyses, the resultant cDNA was diluted 100-fold, and 1 µL was used as the template in a PCR amplification containing 0.5 µL of Q5 High-Fidelity DNA Polymerase (NEB, M0491S), 1x Q5 polymerase reaction buffer (NEB, B9072S), 0.5 uM of forward and reverse primer, 2.5 mM each of dATP (NEB, N0440S), dCTP (NEB, N0441S), dGTP (NEB, N0442S), dTTP (NEB, N0443S) in a 50 µL total reaction volume. The amplification conditions were 98 °C for 30 s and then 25 cycles of: 98 °C for 10 s, 55 °C for 20 s, 72 °C for 10 s with a final 72 °C extension step for 2 min. The products were assayed by gel electrophoresis and their concentrations were measured by Fragment Analyzer HS NGS Fragment Kit (Agilent Technologies Inc., DNF-474-0500).