title: GSM5473883: Cyclin D1 p53-/- Pineoblastoma p90 rep2_rna; Mus musculus; RNA-Seq
library_construction_protocol: For RNA-seq, RNA was extracted from pineal gland tissues collected at day 10, 49 and 90 from Cyclin D1 p53-/- genotype using the RNeasy Plus Mini Kit. Tissues were harvested in RLT Plus Buffer. For ATAC-seq, tissues were homogenized in 1X Homogenization Buffer followed by disruption and nuclei were isolated and counted using Trypan blue staining. 50,000 nuclei were collected per condition. For ChIP-seq, tissues were cross-linked on rotator with 1% formaldehyde for 10 min at RT and subsequently quenched by 0.125M glycine at RT for 5 min with mild rocking. Cross-linked tissues were lysed on ice with cell lysis buffer (containing 1% SDS, 10mM EDTA, 50mM Tris, 1X Protease Inhibitor cocktail and PMSF) followed by nuclear lysis buffer (containing 10mM Tris, 1mM EDTA, 1% SDS, and 1X Prostease Inhibitors and PMSF) to release nuclear chromatin. DNA was then sonicated in nuclear lysis buffer into 200bp-700bp fragments using Diagenode Bioruptor. ChIP-seq library preparation was performed using the Illumina Thruplex Rubicon kit. Sequencing was performed on the Illumina Novaseq S1 (2x50 Paired-end). ATAC-seq libraries were prepared by PCR amplification using  Nextera Index Kit (Illumina, no. 15055289) and the NEBNext High-Fidelity 2× PCR Master Mix (NEB, no. M0541L) with 4–6 cycles. Libraries were quantified by PicoGreenn DNA Quantification assay and sequenced on Illumina NovaSeq S2 using 2x50 paired-end reads in biological duplicate.  Sequencing libraries were prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, Inc; San Diego, CA) with an input of 500ng of total RNA following the low sample procedure. Briefly, ribosomal RNA (rRNA) was depleted from total RNA and the remaining RNA purified, fragmented appropriately, and primed for cDNA synthesis. Blunt-ended cDNA was generated after first and second strand synthesis. Adenylation of the 3' blunt-ends was followed by adapter ligation prior to the enrichment of the cDNA fragments. Final library quality control was carried out by evaluating the fragment size on a DNA1000 chip ran on a 2100 BioAnalyzer (Agilent; Santa Clara, CA). The concentration of each library was determined by quantitative PCR (qPCR) by the KAPA Library Quantification Kit for Next Generation Sequencing (KAPA Biosystems; Woburn, MA) prior to sequencing.  Libraries were normalized to 2 nmol/L in 10 mM Tris-Cl, pH8.5 with 0.1% Tween 20 then pooled evenly. The pooled libraries were denatured with 0.05N NaOH and diluted to 20 pmol/L. Cluster generation of the denatured libraries was performed according to the manufacturer's specifications (Illumina, Inc; San Diego, CA) utilizing the HiSeq PE Cluster Kit v2 chemistry and flow cells. Libraries were clustered appropriately with a 1% PhiX spike-in. Sequencing-by-synthesis (SBS) was performed on a HiSeq2500 utilizing v2 chemistry with paired-end 101-bp reads resulting in an average of 58.9 million paired-end reads per sample. Sequence read data were processed and converted to FASTQ format for downstream analysis by Illumina BaseSpace analysis software, FASTQ Generation v1.0.0.