title: BRB-seq V2 (degraded, 1mn)
organism: Homo sapiens
description: Protocols: hASC were obtained from a fresh lipoaspirate as following. 50 ml of lipoaspirate was washed twice with 40 ml of DPBS Ca+/Mg+ (Gibco, #14040091) in 100 ml syringes and incubated with 0.28 U/ml of liberase TM (Roche, #05401119001(ROC)) for 45 min at 37ºC under agitation. The digested tissue was mixed with 40 ml of CRB (1% human albumin (CSL Behring) in 40 ml of DPBS -/- (Gibco, #14190094)) and shaken vigorously to liberate the stromal cells. The aqueous phase was recovered and centrifuged at 400g for 5 minutes at RT. The cell pellet was resuspended in 15 ml of CRB and filtered through a 100 µm and then 40 µm cell strainer to ensure a single cell preparation, centrifuged and resuspended in Minimum Essential Medium (MEM) alpha (Gibco, #32561037) supplemented with 5% human platelet lysate (Cook Regentec, #G34936) and 50 µg/mL Primocin (InvivoGen, #ant-pm-1). hASCs were cultured in the same media composition until 70-80% confluency and detached using TrypLE Select (Life Technology, 1256311) for passaging. For the adipogenic differentiation, cells at confluence were treated with induction cocktail from Adipogenic BulletKit (Lonza, PT-3004) for 7 days, followed by treatment with maintenance cocktail for another 7 days. Total RNA was isolated using TRIzol (Sigma) and followed by double precipitation with ethanol. BRB-seq novel protocol (see referenced paper - https://www.biorxiv.org/content/biorxiv/early/2018/01/30/256594.full.pdf)