title: GSM8685197: P4 Wild Type scATAC-Seq Replicate 2; Mus musculus; ATAC-seq
library_name: GSM8685197
library_construction_protocol: Animal model  Time-mated wild-type (WT) CD1 mice were obtained from Charles River Laboratories. Transgenic mouse lines were maintained on a mixed C67BL6/CD1 background. The following transgenic mouse lines were used in the study:  1) Foxd1-Cre (B6;129S4-Foxd1tm1(GFP/cre)Amc/J, JAX#012463), 2) Isl1-flox (gift from Dr. Lin Gan, (Pan et al. 2008)), 3) Lhx1-flox gift from Dr. Richard R Behringer (Kwan and Behringer 2002), 4) Nkx2.2-Cre (B6.129S6(Cg)-Nkx2-2tm4.1(cre/EGFP)Suss/J, JAX#026880), 5) Dlx1/2-flox (gift from Dr. Jay Lee, (B. Lee et al. 2018)).  Animals were housed in a temperature-controlled facility with a 14-hour dark/10-hour light cycle and had ad libitum access to food and water. All animal procedures followed the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Johns Hopkins University Animal Care and Use Committee. Tissue Collection and Dissections  Dissection of WT Mice  Tissues were collected from WT mice representing a random but equal mix of males and females across postnatal ages. For embryonic stages, 10-30 embryos were harvested from 3-4 pregnant dams, depending on the gestational age. Brain dissections followed previously established protocols(D. W. Kim et al. 2020). Meninges were removed, and dissections were completed in cold HBSS (#14170112, ThermoFisher Scientific) within 30 minutes for each age group.  Dissection of Mutant Mice  Embryos from mutant mice were collected from a minimum of five pregnant dams. Hypothalamic and prethalamic regions were dissected, and the remaining tissues of embryos were used for rapid genotyping (#4359187, Thermo Fischer Scientific), and grouped by genotype for further analysis. Dissected hypothalamic and prethalamic tissues were stored in HABG buffer (Hibernate-E (#A1247601, Thermo Fisher Scientific) for embryos or Hibernate-A (#A1247501, Thermo Fisher Scientific) for postnatal stages, both supplemented with 2% B-27 Supplement (#17504044, Thermo Fischer Scientific), and 0.5 mM GlutaMAX Supplement (#35050061, Thermo Fisher Scientific) until cell dissociation.   Preparations of cells and nuclei  Cell Dissociation for scRNA-Seq  To prepare samples for scRNA-Seq, tissues were dissociated in Hibernate medium (Hibernate-E Minus Calcium (#HECA500, BrainBits) for embryos or Hibernate-A Minus Calcium (#HACA500, BrainBits) for postnatal stages), supplemented with 2 mg/ml papain (#LS003119, Worthington Biochemical) and 0.5 mM Glutamax. The enzyme mixture was pre-incubated for 15 minutes, then added to tissues for 5 - 30 minutes with 0.1 U/μl RNase inhibitor (N2615, Promega). Toward the end of digestion, 100 μg/ml final DNase I (#4716728001, Sigma-Aldrich) was added. Cells were subsequently centrifuged, resuspended in HABG buffer, filtered through a 40 μm cell strainer (BAH136800040-50EA, Sigma-Aldrich), and resuspended in HABG buffer containing RNase inhibitor (0.5 U/μl). Cell viability and counts were assessed with Trypan Blue staining (#15250061, Thermo Fisher Scientific) and a hemocytometer.  Nuclei Preparation for scATAC-Seq  Nuclei for scATAC-Seq were extracted following post-cell dissociation and purified following a 10x Genomics protocol. Nuclei count and yield were verified with Trypan Blue staining and a hemocytometer.  Nuclei Preparation for snRNA-Seq  For snRNA-Seq, adult mouse brains were rapidly dissected, and 100 um coronal sections were prepared using a mouse brain matrice. Regions spanning the ZI, TRN, and thalamus were microdissected under a dissection microscope. Nuclei were extracted following a 10x Genomics protocol. Sequencing Library Preparation and Data Processing  sc/snRNA-Seq  Libraries for scRNA-Seq and snRNA-Seq were prepared using the 10x Genomics Chromium. Single Cell 3' v3.0 and v3.1 platforms (Table S1), following the manufacturer's protocols. Sequencing was conducted on an Illumina NovaSeq 6000, targeting a depth of 500 million reads per sample. Raw data were processed with the 10x Genomics Cell Ranger Software (v5) and aligned to the mm10 mouse genome reference (refdata-gex-mm10-2020-A).  scATAC-Seq  scATAC-Seq libraries were prepared using the 10x Genomics Chromium Single Cell ATAC v1.1 platform (Table S1), with one-hour tagmentation and 9-10 cycles of PCR. Libraries were sequenced on Illumina NovaSeq 6000, at a target depth of 600 million reads per sample. Raw data were processed with Cell Ranger ATAC (v2) and aligned to the mm10 mouse genome reference (refdata-cellranger-arc-mm10-2020-A-2.0.0).