title: GSM8213786: Immune,Patient 2,Before,24; Homo sapiens; OTHER
library_construction_protocol: The RNA extraction was performed according to the protocol. The GeoMx Digital Spatial Profiler (DSP) uses nucleic acid probes coupled to photocleavable oligonucleotide tags which hybridize to targets in slide-mounted tissue sections. Via UV exposure the oligonucleotide tags are released from discrete regions of interest (ROIs) in the tissue. The UV-cleaved oligos are transferred to a collection plate for Library Pre with Seq Code primers. Later they are quantitated by Illumina Next Generation Sequencing. Results are mapped back to tissue location, yielding a spatially resolve digital profile. Library preparation was performed according to Nanostring GeoMX NGS Manual. A specific thermocycler program is set for the PCR process, including various temperature and time settings for steps like UDG incubation, denaturation, annealing, extension, and final holding. Preparation involves selecting the correct primer plates based on the lab worksheet, thawing them, and mixing them with the GeoMx NGS Master Mix and sample aspirates according to defined volumes. This is followed by aliquoting the master mix, transferring primers, and sample aspirates into a new PCR plate. Post-PCR, products are pooled and undergo AMPure cleanup—a bead-based method for purifying the PCR products. The purified library's quality is assessed through capillary electrophoresis, ensuring the correct amplicon size and absence of undesirable products. Quantification of the library concentration is done using Qubit or qPCR. Finally, the library is stored at -20°C until sequencing, with recommendations provided for pooling multiple sample pools for a sequencing run and adjusting loading concentrations based on the Illumina platform used.