title: GSM2337728: Cohort3 (single-cell RNA-seq); Mus musculus; RNA-Seq
library_construction_protocol: For each mouse, tibiae, femurs, iliac crests, and spines were collected for bone marrow cells.  Isolation and enumeration of different hematopoietic cell types was performed by flow cytometry.  Bone marrow cells harvested from each animal were ACK lysed before antibody staining.  We routinely stain 5x10e7 cells per sample for the stem population, and 1x10e7 cells per sample for each progenitor and mature population.  Lineage cocktail consists of biotinylated B220, CD3e, CD4, CD8a, CD19, CD11b, Gr1, Ter119, CD11c, and NK1.1 antibodies.  Fluorescence conjugated to streptavidin was used to recognize lineage cocktail.  Using the following antibody combinations, we were able to identify hematopoietic subpopulations at the stem-cell level: hematopoietic stem cells (HSCs) (Lineage-Pacific Orange, cKit-APC-Cy7, Sca-PE-Cy7, CD48-APC, CD150-PE-Cy5) For WGBS, purified genomic DNA was fragmented to a size range of 100-400bp, 20-50ng of DNA fragments from each sample were end-repaired and ligated with indexed adapters using the NuGen’s Ovation® Ultralow Methyl-Seq Library Systems.  Adapter-equipped DNA fragments were subjected to bisulfite treatments using the QIAGEN Epitect Bisulfite kit followed manufacture’s recommendation with modifications. Bisulfite converted library DNA was PCR-amplified and sequenced at the Broad Institute Genomics Platform. For RNA-Seq, RNA was extracted from sample, reverse transcribed to cDNA, and was subjected to whole genome sequencing at Partners Healthcare Center for Personalized Genetic Medicine. For single-cell RNA-seq, Single-cells were sorted using a BD FACSAria II (BD Biosciences) into PCR 384 well plates (ThermoFisher Scientific) containing standard lysis buffer. Whole transcriptome amplification was performed using the Smart-seq2 protocol (Picelli et al., 2013), and libraries prepared by Nextera XT (Illumina). Samples were pooled and sequenced on an Illumina Nexseq 500 instrument using a 50 bp paired-end-reads. For ATAC-seq, 10,000 cells of each clone were lysed in 50μl of cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and immediately subjected to a transposition reaction at 37oC for 30 min with 2.5μl transposase enzyme (Illumina Nextera DNA Preparation Kit). Transposed DNA was purified using Qiagen MinElute PCR purification Kit and subjected to library amplification using NEBNext High-Fidelity 2X PCR Master Mix, Invitrogen SYBR Green I Dye, and primers (Table S2). Prior to sequencing, the ATAC-Seq library was assayed for quality using TapeStation and BioAnalyzer instruments, and qPCR.
attributes.tag: GEO Accession
attributes.value: GSM2337728