GoKawiilMice
All experiments involving animals were approved by the Yale University Institutional Animal Care and Use Committee and conducted in compliance with all relevant university, state and federal guidelines. The day of vaginal plug detection was designated as PCD 0.5. The day of birth was designated as PD 0. Fezf2–Gfp (000293-UNC) and Arpp21–Gfp (011848-UCD) transgenic mice were obtained from the Gene Expression Nervous System Atlas55. Cux2-E1–Gfp, Cux2-E2–Gfp and Cux2-E3–Gfp transgenic mice were generated by delivering linearized DNA constructs by pronuclear injection. Three to seven founders from each line were examined for reproducible GFP expression. Zbtb18 KO mice were generated by the Masai laboratory28, and Zbtb18fl/fl mice were generated by the Heng laboratory. Emx1–Cre (The Jackson Laboratory; 005628) mice, Cux2–Cre mice, Neurod6–Cre (also known as Nex1–Cre) mice and CAG–Cat–Gfp (The Jackson Laboratory; 024636) mice were previously generated and described56,57,58,59 (Cre Driver Network at the National Institutes of Health (NIH) Blueprint for Neuroscience Research). Genotyping primers are shown in Supplementary Table 15.
The mice were provided with food and water ad libitum, maintained on a 12-h light/12-h dark cycle, provided veterinary care by the Yale Animal Resources Center and housed in clean groups. All mice used or bred for these experiments were in good health, as approved by the Yale Animal Resources Center and confirmed through regular veterinary monitoring. To maintain genetic diversity, multiple concurrent breeding pairs were maintained and siblings were never mated. Both males and females were used randomly throughout this study.
Although blinding was not relevant for the primary mutant versus control comparison, other aspects ofthe study required careful design to minimize bias. Randomization was implemented during data acquisition. Littermates (WT, HET and KO) were housed together to avoid confounding housing effects on statistical analyses. The experimental cohort comprised age-matched male and female littermates. Including samples from multiple litters further enhanced reproducibility.
Tissue preparation and fluorescence-activated cell sorting
Neocortices from PD 0.5 Fezf2–Gfp, with GFP-expressing neurons enriched in the deep layer (L5–6) predominantly ET neurons (7), and Arpp21–Gfp, with GFP-expressing neurons enriched in predominantly IT neurons55, were dissected under a dissection microscope and minced with a sterile blade. Tails were collected for determining sex and genotyping. Single-cell suspensions from neocortical tissue were prepared by dissociation with a papain-based solution60 and incubated at 37 °C for 15 min with intermittent trituration using autoclaved, fire-polished glass Pasteur pipettes. Cells were then pelleted by centrifugation at 4 °C for 5 min, washed with sterile 1× phosphate-buffered saline (PBS) and filtered through a 40-μm strainer. Cells were then sorted to collect GFP+ cells by fluorescence-activated cell sorting (FACS) using FACSAria II (BD Biosciences) sorter or Beckman Coulter MoFlo sorter. Hibernate solution (Gibco) supplemented with 2% fetal bovine serum (Gibco) was used to collect FACS-sorted cells. Cell preparations were maintained at 4 °C during the entire process. For RNA-seq, immediately after FACS, cells were pelleted by centrifugation at 350g at 4 °C for 10 min, washed with PBS, pelleted and flash-frozen in liquid nitrogen and stored at −80 °C. For ChIP–seq, FACS-sorted GFP+ cells were pelleted by centrifugation at 350g for 10 min at 4 °C, washed with PBS and immediately crosslinked with formaldehyde solution at a final concentration of 1% for 10 min at room temperature. Glycine (AmericanBio) was added at a final concentration of 125 mM, and samples were incubated for 5 min at room temperature to quench crosslinking. Cells were washed again with PBS, pelleted, flash-frozen in liquid nitrogen and stored at −80 °C.
RNA-seq and initial analysis
Total RNA was extracted from FACS-purified cells or neocortical tissue using TRIzol reagent, according to the manufacturer’s instructions. DNase I (Invitrogen) was added to the extracted total RNA, incubated for 15 min at 37 °C to eliminate DNA contaminants and then inactivated according to the manufacturer’s instructions. RNA concentration and integrity were measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific) and TapeStation 2200 (Agilent). Samples with RNA integrity number of 8 or higher were used for subsequent experiments. Libraries were prepared using the TruSeq Stranded Total RNA with Ribo-Zero preparation kit (Illumina), according to the manufacturer’s instructions. Libraries were quality controlled using the TapeStation 2200 (Agilent) and sequenced on the HiSeq 2000 platform (Illumina) at the Yale Center for Genome Analysis (YCGA) to generate 75-bp single-end reads. Sequencing data were quality controlled using FastQC and aligned to the mouse genome (NCBI38/mm10) using TopHat (v.1.0.13) with up to two mismatches61. An average of 40 million uniquely mapped reads were obtained for each sample. Differential expression analysis was performed using the R package DESeq, and principal component analysis was performed using the R package prompt. Differential expression of transcripts was detected using FDR < 0.01.
ChIP–seq and initial analysis
Pooled GFP+ cells from FACS were used for ChIP–seq. A total of 2.5 × 107 cells per condition were crosslinked with a formaldehyde solution (Sigma-Aldrich) at a final concentration of 1% for 10 min at room temperature. L-Glycine (AmericanBio) was added at a final concentration of 125 mM and incubated for 5 min at room temperature to quench the crosslinking. Cells were washed with PBS and then disrupted using lysis buffer I (50 mM HEPES–KOH (pH 7.5), 140 mM NaCl, 1 M EDTA (pH 8.0), 10% glycerol, 0.5% Nonidet P-40 (NP-40), 0.25% Triton X-100 and 1× protease inhibitor) for 20 min at 4 °C and lysis buffer II (200 mM NaCl, 1 M EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 10 mM Tris–HCl (pH 8.0) and 1× protease inhibitor) for 10 min at room temperature. Cells were centrifuged at 300g for 15 min at 4 °C, and pellets were dissolved in 400–600 μl of lysis buffer III (1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0), 10 mM Tris–HCl (pH 8.0), 0.5% sarkosyl and 1× protease inhibitor) before being sheared into 200–500 bp fragments with a sonicator (Bioruptor; Diagenode). Dynabeads Protein G (Invitrogen) was pre-blocked with 5 mg ml−1 of ice-cold bovine serum albumin (BSA) and incubated with 5 μg anti-H3K27ac antibody (Abcam) at 4 °C with constant rotation for 12 h. Chromatin (25 μg) was added to the bead–antibody complex mixture per reaction and incubated with constant rotation for 16 h at 4 °C. Beads were washed with ice-cold radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific) eight times, rinsed with 1× Tris–EDTA (1× Tris–EDTA) solution, eluted by adding 200 μl of ChIP elution buffer (1% SDS; 1× Tris–EDTA) and incubated in a shaker for 20 min at 65 °C. ChIP DNA was incubated for 12 h at 65 °C for reverse crosslinking, treated with RNAse A (Thermo Fisher Scientific; 1 h; 37 °C) and Proteinase K (Sigma-Aldrich; 2 h; 55 °C) and then purified on PCR purification columns. For input control, 5 μg of chromatin from whole-cell extract of each sample was subjected to reverse crosslinking, RNase A treatment (Thermo Fisher Scientific; EN0531) and Proteinase K treatment (Sigma-Aldrich; 3115887001), together with immunoprecipitated samples, and purified using PCR purification columns. DNA amounts were quantified using the PicoGreen assay (Thermo Fisher Scientific; P7589). Immunoprecipitated DNA (5 ng) and input from each sample were used to prepare ChIP libraries with TruSeq ChIP Library Preparation Kit (Illumina; IP-202-1012), according to the manufacturer’s instructions. Libraries were size selected to enrich 300–400 bp size fragments, quality controlled and sequenced on Hiseq 2000 platform (Illumina) (YCGA). Approximately 20–25 million reads were obtained from each sample. FASTA files were mapped to the mouse genome (NCBI37/mm10) using TopHat v.1.0.13 (http://tophat.cbcb.umd.edu/) and Bowtie 2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)62. Peaks were identified using MACS2 (SCR_013291)63.
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