title: GSM4763868: E17-5-9 scATAC-seq; Mus musculus; ATAC-seq
library_construction_protocol: Dissociated cells resuspended in PBS + 0.04% BSA were treated with DNase I prior to nuclei isolation.  Specifically, cells were pelleted, the supernatant removed, and the cell pellet resuspended in DNase Solution (1X TBS, 1X Reaction Buffer with MgCl2 (ThermoFisher Scientific, Waltham, MA), 0.1 U/l DNase I (ThermoFisher Scientific), nuclease-free water).  Cells were then incubated for 5 mins, pelleted, and washed twice with PBS + 0.04% BSA.  The cell suspension was passed through a 40 m Flowmi Cell Strainer (VWR, Radnor, PA), and the cell concentration determined before proceeding to nuclei isolation. For nuclei isolation, up to 1x106 cells were pelleted, followed by removal of the supernatant, and resuspension in chilled 0.1X Lysis Buffer (1 mM Tris-HCl pH 7.4, 1 mM NaCl, 0.3 mM MgCl2, 0.01% Tween-20, 0.01% Nonidet P40 Substitute, 0.001% Digitonin (Sigma-Aldrich, St. Louis, MO), 0.1% BSA (Miltenyi Biotec, Bergisch Gladbach, Germany), and nuclease-free water) for a pre-determined optimal cell lysis time.  Wash Buffer was added to dilute the lysis buffer, then the nuclei were immediately pelleted.  The supernatant was removed, and the isolated nuclei were resuspended in 1X Nuclei Buffer (10x Genomics, Pleasanton, CA).  Finally, nuclei concentration was determined and scATAC-Seq transposition and library construction was immediately performed. scATAC-Seq transposition and library construction was performed according to the manufacturer's protocol using the Chromium Next GEM Single Cell ATAC v 1.1 reagent kit (10x Genomics).  Transposition of single nuclei obtained in the previous step was performed in bulk, followed by capture of the transposed, single nuclei into GEMs (Gel Bead-In-Emulsions) using the Chromium Controller (10x Genomics).  GEM cleanup and library size selection was performed using Dynabeads MyOne Silane beads (10x Genomics) and SPRIselect reagent (Beckman Coulter B23318), respectively.  Sample index PCR was performed for 10 cycles.  Indexed sequencing libraries were quantified by qPCR using the Collibri Library Quantification Kit for Illumina platforms (ThermoFisher Scientific).  Libraries were sequenced on the NovaSeq (Illumina, San Diego, CA) at 2x50bp at a sequencing depth of ~25K reads per nuclei.
attributes.tag: GEO Accession
attributes.value: GSM4763868