GSM9689516: Foxp3Ctrl tdLN, 2; Mus musculus; RNA-Seq
Lymph node and tumour tissues (≤ 5mg) were protected in RNAlater (Sigma; R0901-500ML) at -20°C until being homogenized with CK14 lysing beads in buffer RLT. Total RNA was extracted with the RNeasy® Micro kit (Qiagen; 74034) following the manufacturer's protocol. RNA quality was checked using the TapeStation system (Agilent), only RNA samples with RIN score over 9.0 were sent to Genewiz for sequencing. RNA sequencing libraries were constructed by Genewiz using a standard protocol in which total RNA quality is first assessed, followed by enrichment of polyadenylated mRNA using oligo(dT) magnetic beads or depletion of rRNA for total RNA applications; the resulting RNA is fragmented (typically to ~200 bp) and subjected to first- and second-strand cDNA synthesis, after which fragments are end-repaired, 3' adenylated, and ligated to Illumina-compatible indexed adapters; the adapter-ligated products are then PCR-amplified to enrich for properly ligated fragments, and the final libraries are quality controlled for size distribution and concentration before sequencing on an Illumina NovaSeq 6000 platform.