title: GSM7876410: RPM_4_&_6 weeks tumor_rep2; Mus musculus; OTHER
library_name: GSM7876410
library_construction_protocol: We generated Visium data from two adjacent sections as technical replicates, with each slide containing RPM tumor tissues from at least two individual mice. Visium Spatial Gene Expression slides were prepared with FFPE sections by the Molecular Cytology Core at MSKCC. Tumor samples with a target RIN value > 0.5 were processed for spatial transcriptomics. Probe pairs targeting the whole transcriptome were added to slide capture areas and allowed to hybridize overnight at 50°C. Bound pairs were ligated to one another, then released from the tissue by RNase treatment and permeabilization and captured by oligos bound to the slide. Probe extension and library preparation proceeded using the Visium Spatial for FFPE Gene Expression Kit, Mouse Transcriptome (10X Genomics, 1000337) according to the manufacturer's protocol. After evaluation by real-time PCR, sequencing libraries were prepared with 11-14 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NovaSeq 6000 in a PE28/88 run using the NovaSeq 6000 SP Reagent Kit (100 cycles) (Illumina). FASTQ files from sequencing (NovaSeq) were processed via spaceranger count (version 2.0.0) to align reads to the GRCm38 (mm10) reference genome and generate count matrices for subsequent bioinformatic analyses. An average of 74 million paired reads was generated per sample, corresponding to 37,000 reads per spot.