GSM4569656: E17_FB_RNA-Seq_Ts65Dn; Brwd1+/– _1; Mus musculus; RNA-Seq
RNA: RNA was isolated on RNeasy Microcolumns (Qiagen) following the manufacturer's instructions; ChIP: Hippocampal tissues (unilateral hippocampus from two animals were pooled/biological replicate; Euploid, Ts65Dn and Ts65Dn; BRWD1+/-, 6-8 wk old) were crosslinked, quenched and processed, as previously described. Briefly, samples were washed thoroughly, lysed and sonicated. Samples were then incubated with a custom anti-Smarca2/4 antibody provided by the Crabtree lab (15) (7.5 ug/sample) bound to sheep anti-rabbit M-280 Dynabeads (Invitrogen) on a rotator at 4°C O/N. The following day, immunoprecipates were washed, eluted and reverse-crosslinked in elution buffer O/N. DNA was purified using a PCR purification kit (Qiagen); ATAC: Nuclei Isolation for (Fluorescence Activated Nuclear Sorting) FANS Frozen whole hippocampus was homogenized in cold lysis buffer (0.32M Sucrose, 5 mM CaCl2, 3 mM Mg(Ace)2, 0.1 mM, EDTA, 10mM Tris-HCl, pH8, 1 mM DTT, 0.1% Triton X-100) by douncing 50x in a 10ml dounce homogenizer and filtered through a 40 µm cell strainer to isolate nuclei. The flow-through was underlaid with sucrose solution (1.8 M Sucrose, 3 mM Mg(Ace)2, 1 mM DTT, 10 mM Tris-HCl, pH8) and subjected to centrifugation at 4,000 rpm for 15 minutes at 4°C to remove cellular debris. Pellets were thoroughly resuspended in 500 µl DPBS and incubated in BSA (final concentration 0.1%) and anti-NeuN antibody (1:1000, Alexa488 conjugated, Millipore, cat#: MAB377X) under rotation for 1 hour at 4 °C in the dark. Prior to FANS, DAPI (Sigma cat#: MBD0015) was added to a final concentration of 1 µg/ml. Unstained nuclei and nuclei stained with only secondary antibody served as negative controls. DAPI positive neuronal (NeuN+) nuclei were sorted into tubes using a BD-FACSAria flow cytometer (BD Biosciences) equipped with a 70μm nozzle. RNA-seq and ChIP-seq: Following RNA purifications, libraries were prepared according to Illumina protocols and sequenced on an Illumina HiSeq 4000 sequencer; ATAC-seq: ATAC-seq reactions were performed using an established protocol (16) with minor modifications. Following FANS, 50,000 sorted nuclei were centrifuged at 500 × g for 10 min, 4°C. Pellets were re-suspended in transposase reaction mix [25 μL 2x TD Buffer (Illumina Cat #FC-121-1030), 2.5 μL Tn5 Transposase (Illumina Cat #FC-121-1030) and 22.5 μL Nuclease Free H2O] on ice. Samples were incubated at 37 °C for 30 min and then purified using the MinElute Reaction Cleanup kit (Qiagen Cat #28204) according to manufacturer's instructions. Following purification, library fragments were amplified using the Nextera index kit (Illumina Cat #FC-121-1011), under the following cycling conditions: 72 °C for 5 minutes, 98 °C for 30 seconds, followed by thermocycling at 98 °C for 10 seconds, 63 °C for 30 seconds and 72 °C for 1 minute for a total of 5 cycles. In order to prevent saturation due to over-amplification, a 5 µl aliquot was then removed and subjected to qPCR for 20 cycles to calculate the optimal number of cycles needed for the remaining 45 μL reaction. The additional number of cycles was determined as follows: (1) Plot linear Rn vs. Cycle, (2) Calculate the # of cycle that is corresponded to 1⁄4 of maximum fluorescent intensity. In general, we found adding 4-6 cycles to this estimate yielded optimal ATAC-seq libraries. Libraries were amplified for a total of 13–19 cycles. Following PCR, ATAC-seq libraries were purified and double-end size-selected by (.5x ratio and 1.8x ratio) Ampure XP bead purification (Beckman Coulter cat#; A63881) to remove primer-dimers (<100bp) and large fragments (>1000bp). Library size was quantified using Bioanalyzer High Sensitivity DNA Chips (Agilent technologies Cat#5067-4626). Libraries were quantified by quantitative PCR (KAPA Biosystems Cat#KK4873) prior to sequencing. Libraries were sequenced on NovaSeq 6000 System (Illumina) obtaining 2 x 50 paired-end reads.