Illumina NovaSeq 6000 paired end sequencing; Transcriptional changes in the mammary gland during lactation revealed by single cell sequencing of cells from human milk
Transcriptional changes in the mammary gland during lactation revealed by single cell sequencing of cells from human milk
Non-lactating human breast tissue was donated by participants undergoing elective aesthetic reduction mammoplasty. Single cell suspensions of non-lactating mammary cells were generated using an adaptation of a previously described protocol (Linnemann et al. 2015). Briefly, the freshly collected mammary tissue minced in digestion buffer supplemented with 1 µg/mL insulin which was added together with 800 U/mL collagenase and 100 U/mL hyaluronidase and mixed at 100rpm at 37°C for 3-4 hours. Following digestion, the resulting dissociated organoid fragments were washed twice in buffer and pelleted by 300g centrifugation for 5 minutes prior to cryopreservation.  On the day of library preparation, organoid fragments were gently defrosted in a 37°C water bath for approximately 5 minutes before being treated with Trypsin and dispase to yield a highly viable single cell suspension.  Human milk was freshly collected using a breast pump and diluted in an equal volume of sterile phosphate-buffered saline (PBS) and centrifuged at 870 g for 20 minutes at 20°C. The pellet was washed by removing the supernatant and resuspending in 5-10 mL of cold PBS and centrifuging at 490 g for 5 minutes at 4°C. Following a second wash with PBS, the single cell suspension was ready for library preparation. Approximately 10,000 lactation (n=5) or 10,000 non-lactation (n=3) derived mammary cells per donor were loaded into separate channels of a single Chromium controller (10x Genomics) chip. Consequently, gel bead-in-emulsions (GEMS) were generated using 10X Genomics Single Cell 3' Reagent Kit v3. Sequencing libraries were prepared using Single Cell 3' Reagent Kits v3 (10X Genomics) and then converted using the NovaSeq 6000 S2 Reagent Kit (Illumina).