Surgical samples from diseased lung tissue (n = 3 TB+HIV+; n = 3 TB+; n = 2 non-infected patients) were processed as described in (Ardain et al., 2019 (link)). Briefly, each sample was collected into cold RP-10 (RPMI (Sigma-Aldrich) + 10% FBS), minced, and incubated for 25-30 min at 37°C with digestion buffer containing collagenase D (Sigma-Aldrich), DNase I (Sigma-Aldrich) in RPMI 1640 (Sigma-Aldrich) with 10% FBS (Hyclone). Following incubation, samples were homogenized using a GentleMACS, filtered using a 70 μm metal strainer, and pelleted by centrifugation at 400 g for 5 min. After obtaining the pellet, cells were resuspended in RP-10, passed through another 70μm strainer (Corning), stained with trypan blue, and then counted and diluted for scRNA-seq using Seq-Well S3 (see below).
Corresponding Organization : Harvard Stem Cell Institute
Other organizations :
Massachusetts Institute of Technology, Ragon Institute of MGH, MIT and Harvard, Africa Health Research Institute, University of KwaZulu-Natal, University of Massachusetts Chan Medical School, Aix-Marseille Université, Inserm, Université Côte d'Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire, Helmholtz Zentrum München, German Center for Lung Research, Vanderbilt University Medical Center, Massachusetts General Hospital, Brigham and Women's Hospital, University of California, Berkeley, University of Washington, Dana-Farber Cancer Institute, Fred Hutch Cancer Center, Seattle Children's Hospital, University of Pittsburgh, Children's Hospital of Pittsburgh, University of Sheffield, University College London
Tissue processing (mincing, incubation with digestion buffer, homogenization, filtering, centrifugation, resuspension in RP-10, passage through 70μm strainer, trypan blue staining, counting, dilution for scRNA-seq)
Reagents (RPMI, FBS, collagenase D, DNase I)
controls
Positive control: Not specified
Negative control: Non-infected patients
Annotations
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