title: GSM6467475: Melanoma stereoseq; Mus musculus; RNA-Seq
library_construction_protocol: Libraries for scRNA-seq were created using the 10X Genomics Chromium platform according to the manufacturer's instructions. Library construction was performed with the Chromium Single Cell 3ʹ GEM, Library & Gel Bead Kit v3 (n = 16; 10x Genomics, catalog number 1000092) or the Chromium Single Cell A Chip Kit and 5′ Library & Gel Bead Kit (10x Genomics, catalog number 1000014). We opted for high target recovery (median 5000, range 5000-10000), keeping within the range of optimal input concentration per target recovery, as recommended by the manufacturer. In brief, cells were partitioned into gel bead-in-emulsions (GEMs) at limiting dilutions, where lysis and reverse transcription occurred, yielding uniquely barcoded full-length cDNA from polyadenylated mRNA. GEMs were subsequently broken, and the pooled fraction was amplified and subjected to fragmentation, end repair and adaptor ligation of size-selected fractions.For bulk RNAseq about 500 pg of RNA per sample was reverse-transcribed and amplified using a modified SMARTseq2 protocol (Rambow et al. 2018). Prior to generating sequencing libraries using the NexteraXT kit (Illumina, cat. no. FC-131-10), cDNA profiles were monitored using the Bioanalyzer. Sequencing was performed on a Nextseq500 platform (Illumina, SE75bp). Differential analyses between siPRRX1 and siCtrl samples were executed using the DeSeq2 pipeline. Visium libraries were sequenced on the Illumina Novaseq 6000 instrument (parameters: SP flowcell 100 cycles single reads, lane 1 = 220pM + 1% PhiX / lane 2 = 215pM + 1% PhiX).  For stereo seq experiment, the cDNA library was loaded onto a G400 sequencer (MGI) for sequencing (pair-end, read 1 of 50 bp and read 2 of 100 bp).