title: GSM8879992: MC38 tumor tissue, aPD1 treated, replicate 5; Mus musculus; RNA-Seq
library_construction_protocol: Tumors were collected seven days after the start of treatment and prepared as single cell suspensions using the Mouse Tumor Dissociation Kit by Miltenyi Biotec (#130-096-730), according to the manufacturer's recommendations. Briefly, tumors were weighed and minced, then combined with the enzyme mixture in RPMI for enzymatic digestion and loaded onto a gentleMACS Dissociator for mechanical dissociation. Following dissociation, the samples were filtered through 70-micron and 40-micron cell strainers to remove any remaining large particles and cell clumps and washed with media. Samples were initially treated with TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove DNA contaminants. RNA sequencing libraries were constructed with the NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations. Briefly, enriched RNAs are fragments for 15 minutes at 94 ºC. First strand and second strand cDNA are subsequently synthesized. cDNA fragmetns are end repaired and adenylated at 3' ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR.