title: Epigenetics study of      human breast cancer using miRNA
design: For the identification and relative analysis of miRNAs in breast cancer patients, 5 mL of blood was collected from the cancer diagnosed individuals and their corresponding controls in EDTA tubes. The samples were then subjected to density gradient centrifugation and peripheral blood mononuclear cells (PBMC) were isolated and processed for further study. RNA was isolated from the PBMCs using miRNA easy kit (Qiagen, Gmbtt Germany) in accordance with the manufacturer's instructions. The yield of RNA was checked by Nanodrop (Bio photometer plus) and Fluorimeter (Qubit4) and the samples were stored at -80C.RNA samples were pooled for each age group and a consecutive sample per group was obtained. This was done as an attempt to eliminate biological variations resultant of differential gene expressions between samples. Pooling for the patient and cancer samples was carried out alike and total of 6 samples- 3 for study group and 3 for control group, were obtained (Table 1). The pooled RNA samples were measured using the Qubit BR Assay Kit (Invitrogen cat# Q10211) while the purity was checked with QIAxpert and RIN.The extracted RNA was used to prepare sRNA libraries using the NEB Next Multiplex Small RNA Library Prep kit protocol (NEB, #E7560S) and in accordance with the manufacturer's protocol. Qubit High Sensitivity Assay (Invitrogen, Cat# Q32852) was employed for the quantification of the same. The libraries were then pooled and sequencing was carried out on the Illumina HiSeq 2500