title: Sample 10
organism: Homo sapiens
description: Protocols: Fresh lipoaspirates were washed twice with DPBS Ca+/Mg+ (Gibco #14040091) in 100 ml syringes (VWR International #720-2528) (50 ml lipo-aspiratee per 40 ml DPBS Ca+/Mg+) and incubated with 0.28 U/ml of liberase TM (Roche #05401119001(ROC)) for 45 min at 37°C under agitation. The digested tissue was mixed with 1% human albumin (CSL Behring) in DPBS -/- (Gibco #14190094) (50 ml lipo-aspiratee per 40 ml 1% human albumin in DPBS -/-) and shaken to liberate the stromal cells (procedure performed twice). The aqueous phase was recovered and centrifuged (all centrifugations performed at 400g for 5 min at RT). The cell pellet was resuspended in 15 ml remaining buffer and filtered through a 100 µm and then 40 µm cell strainer to ensure a single cell preparation, centrifuged and resuspended in 5 ml of 5% human albumin (CSL Behring). The viability and the number of nucleated cells in the obtained cells suspension was determined using a Nucleostainer, after which a red blood cell lysis was performed using VersaLyse solution (Beckman Coulter #A09777) according to producer's recommendations. The isolated single cell suspension was diluted to 1 * 10^7 cells/ml with FACS buffer (DPBS -/-) with 1% human platelet serum (Cook Medical #G34936 PL-NH-500) and the following fluorophore-conjugated antibodies were added: anti-human CD31-AF488, anti- human CD45-AF488 for selecting the Lin- population, and anti-human CD142-PE (all BioLegend #303110, #304017 and #365204 respectively) for separating CD142+ (F3+) and CD142- (F3-) populations. 7-Aminoactinomycin D (7-AAD) (Beckman Coulter #A07704) was used for assessing viability and Syto40 (Molecular Probes #S11351) for discerning nucleated cells. The cells were sorted directly into flat bottom microscopy-adapted cell culture plates in high glucose MEMalpha medium (Gibco #32561037) supplemented with 5% human platelet serum and 50µg/ml Primocin (InvivoGen #ant-pm-1). The cells at confluence were treated with induction cocktail (high glucose DMEM, 10% FBS, 50µg/ml Primocin, 0.5 mM IBMX, 1 µM dexamethasone, 1.7 µM insulin) for 7 days, followed by maintenance cocktail treatment (high glucose DMEM, 10% FBS, 50µg/ml Primocin, 1.7 µM insulin) for another 7 days. mRNA was isolated and transcribed into cDNA using the MultiMACS cDNA Synthesis Kit (Miltenyi #130-094-410) or collected into Tri-Reagent (Molecular Research Center #TR118). Direct-zol RNA kit (Zymo Research #R2052) was used to extract RNA, followed by reverse transcription using SuperScript VILO cDNA Synthesis Kit (Invitrogen). Bulk mRNA sequencing was performed in a barcoded fashion, using a time, labor and cost-efficient preparation of a single Nextera-compatible cDNA library containing up to 96 (and potentially more) different RNA samples (Alpern et al., in preparation). The protocol is adopted from SCRB-seq, developed for single cell transcription profiling, with several important modifications. For the current series of experiments, 50 ng of total RNA from each sample was reverse transcribed in a 96-well plate using Maxima H Minus RT (Thermo) with individual oligo-dT primers, featuring a 6 nt long multiplexing barcode, and template switch oligo (Microsynth). Specifically, each oligo-dT primer is biotinylated and has the following structure: 5'- ACACTCTTTCCCTACACGACGCTCTTCCGATCT[BC6] [N15] [T30] VN-3', where, [BC6] = 6 nt barcode specific to each well; N15 stretch of random nucleotides forming a Unique Molecular Identifier (UMI). Thus, for each well, we have generated a unique combination of barcodes and UMIs to identify each well (sample) and transcript. Next, all the samples were pooled together, purified using the DNA Clean and Concentration kit (Zymo), and treated with exonuclease I (NEB). The full-length cDNA library was amplified using a single primer and purified with AMPure beads (Beckman Coulter). The sequencing library was prepared by tagmentation of 10 ng full length cDNA with an in house made Tn5 transposase at 55çC for 9 minutes. Tagmented DNA was purified with the DNA Clean and Concentration kit and PCR amplified using NEBNext High-Fidelity 2X PCR Master Mix (NEB) with an i7 adapter identical to Illumina Nextera and custom i5 (Microsynth). The PCR reaction was then purified twice with AMPure beads and the average fragment size of the library was evaluated using a Fragment Analyzer (Advanced Analytical) prior to paired-end sequencing with NextSeq 500 (Illumina).