GSM8244461: 4-P007-TLK1_S4; Homo sapiens; RNA-Seq
Genomic DNA was subjected to 35 cycles of PCR amplification using Q5 High-Fidelity DNA Polymerase (New England Biolabs) with primers that allow for the amplification of the region containing the variants of interest. PCR products were run in a 2% agarose gel and purified using the Nucleospin Gel and PCR clean-up kit (Macherey-Nagel) following manufacturer's instructions. Briefly, bands were carefully excised with a clean scalpel, weighed and incubated at 50°C for 10 minutes with the corresponding amount of NTI Buffer. Next, DNA was bound at a silica membrane by placing it into a column provided by the kit. The membrane was then washed twice with NT3 buffer and dried by incubating at 70°C for 3 minutes. DNA was eluted in NE buffer and quantified using a DS-11 Spectrophotometer (DeNovix). PCR products were sequenced using a NextSeq (Illumina). DNA libraries were prepared using the Nextera XT DNA Library Prep kit (Illumina) following manufacturer's instructions. Sequencing of PCR amplicons was carried out using the MiSeq Nano kit sequencing system (Illumina) with 150 bp paired-end (PE) reads.