Illumina HiSeq 2500 paired end sequencing; Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients
Tumor invasion in draining lymph nodes is associated with Treg accumulation in breast cancer patients
niLN_Treg_401_p
CD4+CD45RA-CD25- (Memory CD4+ Tconv), CD4+CD45RA-CD25high (Memory Treg), were sorted by flow cytometry in a BD FACS ARIA II cell sorter, with a purity of 98-99%. Cells were collected in TCL buffer (Qiagen) with 1% of b-mercaptoethanol and stored at -80 until subsequent analysis.  RNA was isolated using a Single Cell RNA purification kit (Norgen),including RNase-Free DNase Set (Qiagen) treatment. RNA integrity number was evaluated with an Agilent RNA 600s0 pico kit.  Retrotranscription was carry out with SMARTv4 low input kit (Takara). Barcoded Illumina compatible libraries were generated from 5-10ng of DNA of each sample, using Nextera XTP reparation Kit.