Illumina HiSeq 2500 sequencing; RNA-seq of breast milk-derived exosomes from type 1 diabetic mothers compared to healthy controls
RNA-seq of breast milk-derived exosomes from type 1 diabetic mothers compared to healthy controls
Exosomes were isolated by serial ultra-centrifugation method. Briefly, 50 ml milk sample was diluted with an equal volume of 1X PBS (pH7.4) and centrifuged at 300 x g for 10 min at 4ºC to pellet the cells. Supernatant was further centrifuged at 3000 X g for 10 min at 4ºC followed by sequential filtrating of the supernatant through 1.2 µm, 0.8 µm, 0.45 µm and 0.2 µm filters (VWR®). Resultant filtrate was centrifuged at 16500  X g for 45 min at 4ºC and supernatant was again filtered using 0.2 µm filter followed by centrifugation at 12000 X g for 70 min at 4ºC to pellet exosomes. Exosome pellet was resuspended in 300 µl of 1X PBS.  Total RNA was purified from the isolated exosomes using miRNeasy kit (Qiagen) according to the manufacturer's instructions.  All libraries were prepared using the Illumina TruSeq Small RNA protocol following the manufacturer's instructions with 12 cycles of PCR amplification after ligation of the 3′ and 5′ adapters
Experimental Factor: disease
normal