Novaseq sp

RNA-sequencing was performed on 3 biological replicates of KD, DBD and DBD+. Total RNA were extracted using RNeasy Extraction Kit and submitted to the Nationwide Children’s Hospital Institute for Genomic Medicine for RNA quality measurement, library preparation, and sequencing. Briefly, cDNA libraries were prepared from total RNA with TruSeq Stranded mRNA Kit (Illumina 20020594) and sequenced on Illumina NovaSeq SP to generate 150-bp paired-end reads. We used in-house RNA-sequencing pipeline to process and analyze the data. Low-quality reads (q < 10) and adapter sequences were trimmed to align to hg19 genome using STAR (Dobin et al., 2013 (link)). After alignment, the reads were counted and differential analysis performed using DESeq2 (Love et al., 2014 (link)).

EpdSCs were collected by FACS as described above directly into TRIzol LS (Invitrogen). RNA libraries were generated using SMARTer RNA kit for low-input RNA-seq. Libraries were sequenced on Illumina NovaSeq SP. Raw FASTQ files were trimmed of barcodes using Skewer (v.0.2.2) and transcript abundance quantified using Salmon (v.1.4.0) with a modified GENCODE transcript index (version GRCm38 release M24) to include TRE-Sox9. Gene level counts and transcripts per million (TPM) were calculated using the Tximport (v.1.12.3) package in R (v.3.6.1). For hair placode RNA-seq data, after generating the raw counts, differentially expressed (DEG) gene list was generated with DESeq2 (v.1.16.1).

ATAC-seq library preparation was performed on FACS-sorted mouse AMs (200,000 cells per sample) using the ATAC-seq kit from Active motif (#13150) according to the manufacturer’s protocol. The purified DNA was amplified for 10 cycles using indexed primers and size-selected using SPRI bead solution. A quality control (QC) was performed to verify the size distribution of the PCR-enriched library fragments. To this end, aliquots of the DNA libraries were analyzed on a TapeStation, and quantification of the libraries was done using the Qubit. The bar-corded amplicons from ATAC-Library preparation were sequenced in a NovaSeq SP (Illumina) under a 2 × 150 pair-ended format. Reads were quality filtered according to the standard Illumina pipeline, de-multiplexed, and fastq files were generated (GEO accession no. GSE231219).

Data were collected utilizing the following open-source or commercially available software programs: BD FACSDiva (version 8.0.2), BioRad Image Lab (version 6.0.1, build 34), BioRad CFX Manager (version 3.1). ATAC-seq data was generated by Illumina Novaseq SP. RNA-seq was performed by Azenta Life Sciences (formerly Genewiz). Analyses and/or manuscript preparation were conducted using the Microsoft Office Suite, version 16.43 (including Microsoft Word, Microsoft Excel, and Microsoft Powerpoint), BD FlowJo (version 10.8.1), and open-source software, including tools available on Galaxy (http://usegalaxy.org): Bowtie2 (2.4.4), MACS2, Integrative Genomics Viewer (version 2.9.4), Morpheus (Broad Institute), VolcanoseR (https://huygens.science.uva.nl/VolcaNoseR/)^{75 (link)}, and ClustVis (https://bio.tools/clustvis). All statistical analyses were performed using the GraphPad Prism software (version 9.3.1). Data preparation for this manuscript did not require the use of custom code or software. Supplementary Fig. 16 was generated using BioRender (https://biorender.com/).

ChIP was performed as previously described (Lee et al., 2006 (link)). In brief, 150 μl of ovaries were dissected into ice-cold PBS, crosslinked with 1.8% formaldehyde in PBS for 10 min at room temperature, quenched with Glycine, rinsed in PBS and flash frozen in liquid nitrogen after removing all PBS. Frozen ovaries were disrupted in PBS using a dounce homogenizer, centrifuged at low speed and the pellet was resuspended in lysis buffer. For ChIP from OSCs 5–10 million cells were crosslinked, quenched, and lysed. Sonication (Bioruptor) resulted in DNA fragment sizes of 200–800 bp. Immunoprecipitation with specific antibodies was done overnight at 4 °C in 350–700 μl total volume using 1/3 to 1/4 of chromatin per ChIP (antibodies are listed in Key Resource Table). Then, 40 μl Dynabeads (equal mixture of Protein G and A, Invitrogen) were added and incubated for 1 hr at 4°. After multiple washes, immuno-precipitated protein-DNA complexes were eluted with 1% SDS, treated with RNAse-A, decrosslinked overnight at 65 °C, and proteins were digested with proteinase K before clean-up using ChIP DNA Clean & Concentrator columns (Zymo Research). Barcoded libraries were prepared according to manufacturer’s instructions using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), and sequenced on a HiSeqV4, NextSeq550, or NovaSeqSP (Illumina).