 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 01, 2017 |
| Title |
Ntsr1_L6_500cell_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
Ntsr1_L6_500cell
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
mouse_id: 178043
age (days): 55
cre driver: Ntsr1-Cre
reporter: Ai14(TITL-LSL-tdTomato)
broad class: Glutamatergic
cortical layer: L6
tissue/cell type: Visual cortex
|
| Growth protocol |
Animals were provided food and water ad libitum and were maintained at a regular 12 h day/night cycle at no more than 5 adult animals per cage. Animals were maintained on the C57BL/6J background.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We used an isoflurane chamber to anesthetize adult male mice (P56 ± 3), decapitated them, removed the brains, and transferred them immediately to freshly prepared, ice-cold artificial cerebrospinal fluid (ACSF: 126 mM NaCl, 20 mM NaHCO3, 20 mM dextrose, 3 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 2 mM mgCL2, 50 μM DL-AP5 sodium salt, 20 μM DNQX, and 0.1 μM tetrodotoxin, mixed then bubbled with 95% O2/5% CO2 carbogen gas). We sectioned the brains to generate 400 μm–thick sections using a Leica VT1000S vibratome with a chilled chamber, and immediately transferred the slices to a bubbled ACSF-containing chamberat room temperature. Individual slices of interest were microdissected in a Petri dish while submerged in ACSF under a fluorescence dissecting microscope. Dissected tissue was transferred to a microcentrifuge tube containing ACSF with 1 mg/mL pronase (Sigma, Cat#P6911) for 70 min at room temperature. After incubation, with tissue pieces settled at the bottom of the tubes, ACSF with pronase was exchanged twice with ACSF containing 1% fetal bovine serum (FBS). We next dissociated the tissue into a single-cell suspension by trituration through Pasteur pipettes with polished openings of 600 μm-, 300 μm-, and 150 μm-diameter. 500 cells were sorted into a well of an 8-well PCR strip containing 2 μL ACSF on a BD FACSAriaII SORP with a 130 μm nozzle at 10 psi sheath pressure, and in the single-cell sorting mode. We excluded dead cells by labelling with DAPI (DAPI*2HCl, Life Technologies Cat#D1306) added to the single-cell suspension at 2 ng/mL. We retained only cells that had high tdTomato fluorescence and low DAPI labeling.
Immediately after FACS collection, cells were lysed with 25 µL Lysis Buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.1 % IGEPAL CA-630). Nuclei were pelleted by centrifugation at 500 x g for 30 minutes at 4 °C in a refrigerated microcentrifuge. After pelleting, the supernatant was removed and cells were resuspended by repeated pipetting in 25 µL Reaction Mix (12.5 µL Nextera TD Buffer, 2 µL Nextera TD Enzyme, and 10.5 µL water). Samples were then tagmented by incubation at 37 °C for 1 hour on a heat block. After tagmentation, the reactions were stopped with addition of 5 µL Cleanup Buffer (900 mM NaCl, 300 mM EDTA), 2 µL 5% SDS, and 2 µL Proteinase K and incubation at 40 °C for 30 min. Tagmented DNA was purified using AMPure XP beads at a ratio of 1.8:1 beads to reaction volume, with a final resuspension volume of 11 µL. For indexing and amplification, we added 15 µL KAPA HotStart Ready mix (Kapa Biosystems, Cat# KK2602) and 2 µL each of Nextera i5 and i7 indexed amplification primers. These samples were incubated at 72 °C for 3 minutes, then PCR amplified as follows: 95 °C for 3 min; 9 cycles of 98 °C for 20 sec, 65 °C for 15 sec, and 72 °C for 15 sec; 72 °C final extension for 1 min. Samples were then purified using AMPure XP as above, re-amplified for additional 9 cycles under the same conditions, and then purified once more as before using Ampure XP beads to produce 11 µL final volume. Library quality and quantity were assessed using 1 µL of the final, purified DNA on a BioAnalyzer High Sensitivity DNA chip. For mES cell populations, ES cells grown on mouse embryonic fibroblast (MEF) feeders were passaged onto gelatin plates to dilute/remove feeder cells, and they were made into single-cell suspensions by treatment with Trypsin-EDTA (Thermo Fisher Scientific, Cat#25300054). Cells were washed twice in PBS with 1% FBS, then were resuspended in PBS with 1% FBS and 2 ng/mL DAPI. 500-cell populations of DAPI-negative ES cells were sorted into individual wells of an 8-well PCR strip containing 2 uL of PBS.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
ntsr1_3
|
| Data processing |
library strategy: ATAC-seq
Reads in FASTQ format were aligned to GRCm38 (mm10) using Bowtie v1.1.0 (Langmead et al., 2009) with settings –m 1 –X 2000 –chunkmbs 256 in paired-end mode.
Unaligned reads were processed with the Trim galore wrapper for Cutadapt (Martin, 2011) to remove Nextera primer sequences (settings: --nextera –paired –three_prime_clip_R1 1 –three_prime_clip_R2 1), and were then aligned again using Bowtie (settings –m 1 –X 2000 -3 1 –chunkmbs 256).
The Samtools collection (Li et al., 2009) was used to sort, remove PCR duplicates (rmdup), index BAM files, and calculate library statistics (flagstat). We used CollectInsertSizeMetrics.jar from Picard v1.110 (Broad Institute 2015) to analyze fragment size statistics, and preseq v0.1.0 (Daley and Smith, 2013) to analyze library sequencing saturation.
To downsample BAM files to 3.2M reads, the data were sorted by name instead of location using Samtools sort in SAM format, then R sample() was used to select random read pairs, and a custom Perl script then filtered the selected reads. Samtools view was then used to convert files back to BAM format.
After downsampling, aligned reads were analyzed for peak and region enrichment of open chromatin using HotSpot v4.1 (John et al., 2011) with default settings (FDR < 0.01).
DiffBind v1.16.3 (Stark and Brown, 2011,Ross-Innes et al., 2012) was used to calculate merged peak locations, based on the outer boundaries of overlapping peaks from all cell lines analyzed, and peak accessibility scores for each replicate were calculated using DiffBind with the default setting DBA_SCORE_TMM_MINUS_EFFECTIVE, which uses trimmed mean of M-values (TMM) normalization (Robinson and Oshlack, 2010) built into the edgeR package (Robinson et al., 2010) using read counts minus control (genomic) read counts and full library size.
For overall views of aligned data from each cell class, we built merged BAM files using samtools merge and sort.
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak: chr, start, and end coordinates from HotSpot peak results
Supplementary_files_format_and_content: broadPeak: chr, start, and end coordinates form HotSpot region results
Supplementary_files_format_and_content: bed: merged peak locations in BED format as calculated by DiffBind
Supplementary_files_format_and_content: tmmScores: A matrix of TMM scores for each merged peak in each sample as calculated by DiffBind
|
| |
|
| Submission date |
Oct 03, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Lucas T Gray |
| E-mail(s) |
lucasg@alleninstitute.org
|
| Organization name |
Allen Institute for Brain Science
|
| Department |
Molecular Genetics
|
| Lab |
Tasic
|
| Street address |
615 Westlake Ave N
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE87548 |
Layer-specific ATAC-seq of the neurons of adult mouse visual cortex defined by Cre-driver lines |
|
| Relations |
| BioSample |
SAMN05858792 |
| SRA |
SRX2207653 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2333640_ntsr1_rep3_3.2M.broadPeak.gz |
846.7 Kb |
(ftp)(http) |
BROADPEAK |
| GSM2333640_ntsr1_rep3_3.2M.narrowPeak.gz |
779.8 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
