GSM8465156: PBMC, habitual sleep, participant 1, GEM well/lane 1; Homo sapiens; RNA-Seq
At baseline of each study phase, participants came to the laboratory to complete various procedures and receive their sleep assignment. Participants returned at least every other week for additional assessments, most of which are beyond the scope of this study. At each of these study visits, sleep data were reviewed with a trained study staff member to assess compliance. At study visit week 5, an evening blood draw (target time: 15:00–17:00) was collected. Peripheral blood was drawn into an EDTA tube from an antecubital vein by a trained phlebotomist. The amount of blood collected and time of draw were recorded at all visits. Whole blood was subsequently processed for collection and preservation of the viability of PBMCs. As described previously7, whole blood was transferred to a 50 ml conical tube (Corning) and PBS diluted to 1× (Thermo Fisher Scientific) was added to the blood to a final volume of 35 ml. The solution was then carefully underlaid with 15 ml of Ficoll-Paque PLUS (GE Healthcare) and spun for 30 minutes at 23°C and 1,800 rpm with the centrifuge brake off (Eppendorf 5810R; Eppendorf). The resulting white blood cell layer was aspirated and transferred to a new conical tube; PBS was added to a final volume of 50 ml and then spun for 10 minutes at 4°C (1800 rpm, brake on). After spinning, the white blood cell pellet was resuspended in freezing media (20% FBS RPMI 1640 [Thermo Fisher Scientific] with 10% DMSO [Sigma Life Sciences]) and transferred into separate 1.5 ml cryovials. The cryovials were frozen at a controlled rate of −1°C/min (Mr. Frosty; Thermo Fisher Scientific) to maintain viability of cells and were then transferred to liquid nitrogen for storage until flow cytometry analysis. Neutrophils are not included in the leukocytes isolated by Ficoll-Plaque PLUS gradient as they are pelleted and discarded. Library was constructed according to manufacturer's protocol (10x Single Cell 3' v3 and Cell Plex)