Macs smartstrainer

Mice were treated with saline, PDGF-BB, bleomycin, or PDGF-BB + bleomycin, as described above. Twenty-four hours after the third injection, mice were euthanized and 12-mm skin biopsies were collected and processed into single-cell suspensions as previously described [18 (link)]. Briefly, excess fat first removed from the underside of the skin biopsy and the remaining tissue was cut into small pieces. Skin tissue was digested in RPMI 1640 supplemented with 10 mM HEPES, 2.6 units/ml Liberase TM (Roche), 100 μg/ml DNase I (Roche), 0.5 mg/ml Hyaluronidase (Sigma), and 1X Penicillin/Streptomycin solution (Invitrogen) for 1 hour at 37°C with gentle agitation. After enzymatic digestion, the cell suspensions were placed in gentleMACS C tubes (Miltenyi) and further disrupted using a gentleMACS homogenizer (Miltenyi) before filtration using a 100 μm MACS SmartStrainer (Miltenyi) for FACS staining. Following a blocking step with FcBlock (BD Biosciences), cell surface protein expression was analyzed using the following specific fluorescently conjugated mAbs: CD45 (30-F11), CD11b (M1/70), Ly6C (HK1.4), B220 (RA3-6B2), CD317 (927), F4/80 (CI:A3-1), CD31 (390), and PDGFRβ (APB5). All antibodies were purchased from BioLegend (San Diego, CA).

Single-cell suspensions of thymus, spleen and lymph nodes were prepared by mechanically dissociating each individual organ through a 30-μm nylon mesh MACS SmartStrainer (Miltenyi Biotec) into cold fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) with 2% foetal calf serum (FCS)). Splenocytes were subjected to red blood cell lysis before resuspension into FACS buffer. Lung and liver tissues were perfused with 10 ml PBS immediately after mice were sacrificed. Lungs were repeatedly sheared into small pieces prior to enzymatic digestion with 3 mg/ml collagenase type III (Worthington Biochemical Corporation) supplemented with 2% FCS, at 37 °C for 60 min. Perfused livers were mechanically dissociated through 70-μm nylon mesh MACS SmartStrainers and then purified for lymphocytes with a 33% isotonic Percoll (GE Healthcare) gradient.

A total of 200 mg tumor tissue was dissected into small pieces and digested using a Tumor Dissociation Kit and GentleMACS Dissociator (Miltenyi Biotec). The digested tumor tissue was filtered through a 70 μm MACS SmartStrainer (Miltenyi Biotec), and the mass remaining on the filter was dissociated using Accutase (Nacalai Tesque) to obtain a single-cell suspension. The scRNA-Seq library was prepared using a BD Rhapsody Single-Cell Analysis system (BD) following the manufacturer’s instruction. Briefly, 20,000 cells were loaded onto the BD Rhapsody microwell cartridge, and cDNAs were synthesized using a BD Rhapsody Whole Transcriptome Analysis Amplification Kit. Gene expression libraries were sequenced on the Illumina NextSeq 550 platform (Illumina) with paired-end reads (read 1, 75 bp; index 1, 8 bp; read 2, 75 bp). Sequencing data were processed using BD Rhapsody Analysis pipelines on the Seven Bridges Genomics platform and converted to the gene expression count matrix.

After 5 days of incubation of GFAP-CreERT2;p53^flox/flox;LSL-tdTomato primary postnatal or adult astrocytes in media supplemented with EGF and FGF and 4OHT, cells were harvested by trypsinisation. Cells were washed and filtered through a 30μm MACS SmartStrainer (Miltenyi), as described in the 10X Genomics manual. Cells were counted with a haemocytometer using trypan blue for exclusion of dead cells. The volume was adjusted to cell concentration of 700-1200 cells/μl, with >80% viability. For the postnatal astrocytes, two replicates were performed from two independent astrocyte preparations, A19 and A26, processed on different days. Gel Bead-In-EMulsions (GEMs) and library preparation was performed as per the 10x Genomics Single Cell 3’ v2 reagent kit protocol. The two libraries from postnatal astrocytes preps were sequenced on a single lane of Illumina HiSeq 2500 (Paired End 2 x 100bp) and de-multiplexed and analysed using CellRanger v3.1.0 and bcl2fastq v2.20.0.
For the adult in vitro astrocytes experiment, library preparation from single cells was performed as per the 10X Genomics Chromium Next GEM Single Cell 3' Kit v3.1 and sequenced on an Illumina Novaseq 6000 Instrument (Paired End, 2x 150bp).

Frozen subcutaneous adipose tissue was minced over dry ice and transferred into ice cold lysis buffer consisting of 0.1% NP-40, 10mM Tris-Hcl, 10 mM NaCl, and 3 mM MgCl2. After a 10-minute incubation period, the lysate was gently homogenized using a dounce and filtered through a 70 μm MACS smart strainer (Miltenyi Biotec #130-098-462) to remove debris. Nuclei were centrifuged at 500 g for 5 minutes at 4°C and re-suspended in wash buffer consisting of 1X PBS, 1.0% BSA, and 0.2 U/μl RNase inhibitor. We further filtered nuclei using a 40 μm Flowmi cell strainer (Sigma Aldrich # BAH136800040) and centrifuged at 500 g for 5 minutes at 4°C. Pelleted nuclei were re-suspended in wash buffer and immediately processed with the 10X Chromium platform following the Single Cell 3′ v2 protocol.
We used Cell Ranger [62 (link)] to build a pre-mRNA alignment reference based on the reference gencode 19 and estimate the UMIs in each cell. As the quality control, we excluded the cells that had <300 genes expressed and kept only the genes that were expressed in at least 3 cells. Then we used Seurat [63 (link)] to simultaneously cluster all the qualified cells from the 6 individuals. We identified 8 clusters and 697 signature genes (S10 Table) that have a higher expression in one of the clusters over the others.