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Sample GSM7018740

Query DataSets for GSM7018740

Status

Public on Jun 07, 2023

Title

SCHW00028

Sample type

SRA

Source name

brain

Organism

Mus musculus

Characteristics

tissue: brain
cell type: Dbh-Cre; Ai14 Neuron

Extracted molecule

total RNA

Extraction protocol

Fresh brain tissue was placed in ice-cold aCSF (2.5 mM KCl, 7 mM MgCl2, 0.5 mM CaCl2, 1.3 mM NaH2PO4, 110 mM choline chloride, 25 mM NaHCO3, 1.3 mM sodium ascorbate, 20 mM glucose, 0.6 mM sodium pyruvate, bubbled in 95% O2 /5%CO2). 200 μm coronal slices that included the LC were collected. Slices were transferred to a 32°C holding chamber containing oxygenated aCSF, where they recovered for at least 1 hour. tdTomato-expressing LC neurons were manually extracted from the slice using glass pipettes pulled from borosilicate glass (World Precision Instruments) to create an ~50 µm tip. The ends of glass pipette tips containing picked cells were snapped into individual wells of a 96 well plate containing 4µl lysis buffer: 0.1µl RNase inhibitor (Thermo Scientific), 0.1µl ERCC RNA spike-in (Invitrogen), 0.02µl 10% Triton (Sigma), 1µl 10mM dNTP (Invitrogen), 0.1µl 100µM dT (IDT; 5'-AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN), and 2.68µl H2O.
Samples underwent primer annealing (72℃ for 3min) before placing on ice. Next, 6µl reverse transcription reagent was added to each sample: 0.14µl H2O, 0.25µl RNase inhibitor, 2µl 5X First Strand Buffer (Clontech), 0.5µl 100mM DTT (Invitrogen), 2µl 5M Betaine (Sigma), 0.06µl 1M MgCl2 (Invitrogen), 0.1µl 100µM TSO (Qiagen; 5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G), 0.95µl SmartScribe enzyme (Clontech). Samples were incubated at 42℃ for 90min, 70℃ for 5min, then held at 4℃. DNA preamplification solution (15µl) was next added to each sample: 2.1375µl H2O, 12.5µl 2X Kapa HiFi Hot Start Ready Mix (Kapa Biosystems), 0.25µl 10µM IS_PCR Primer (IDT; 5'-AAGCAGTGGTATCAACGCAGAGT), 0.1125µl Lambda Exonuclease (NEB). Samples were incubated at 37℃ for 30min, 95℃ for 3min, 21 cycles of [98℃ for 20s, 67℃ for 15sec, 72℃ for 4min], 72℃ for 5min, and then held at 4℃. DNA cleanup, quantification, dilution, tagmentation, and barcoding were performed by the Hartwell Center at St. Jude Children’s Research Hospital using standard methods.
Smart-seq2

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Data processing

Sequences were de-multiplexed using bcl2fastq and aligned to mouse mm10 genome (with Cre and tdTomato genes added).
Paired-end reads from Illumina were trimmed for adapters by cutadapt(v1.15) and aligned to mm9 (MGSCv37, NCBI) by STAR(v2.5.2b, parameters "--outFilterType BySJout --outFilterMultimapNmax 20 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 999 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --outFileNamePrefix SCHW00296. --outSAMstrandField intronMotif --genomeLoad NoSharedMemory --outSAMattributes NH HI NM MD AS nM XS --outSAMunmapped Within KeepPairs --outSAMtype BAM SortedByCoordinate --twopassMode Basic --outFilterScoreMinOverLread 0.33 --outFilterMatchNminOverLread 0.33 --outReadsUnmapped Fastx").
LiftOver was performed on GENCODE(vM9) gene database (from mm10 to mm9) by CrossMap(v0.1.5) to improve gene annotation.
Aligned reads were then counted by HTSEQ (v 0.6.1p1) for genes annotated in GENCODE database. Trimmed reads were also run through FastQ Screen(v0.9.5) against Human, Mouse, Adapters, Univec databases (Dec 2016) to check for contamination and doublets. Samples with larger unique hits to human over mouse genomes were considered putative doublets and excluded from analysis. We further excluded samples with less than 1M reads and those that did not express Dbh.
Assembly: mm9
Supplementary files format and content: raw counts table and quality control matrix

Submission date

Feb 01, 2023

Last update date

Jun 07, 2023

Contact name

Beisi Xu

E-mail(s)

beisi.xu@stjude.org

Organization name

St Jude Children's Research Hosipital

Department

Center for Applied Bioinformatics