GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM2413854

Query DataSets for GSM2413854

Status

Public on Nov 20, 2017

Title

PV-70

Sample type

SRA

Source name

cortex

Organism

Mus musculus

Characteristics

strain: c57bl6, CD-1 mix
age: postnatal day >p21
genotype: PV-Cre x Rosa-tdTomato
position: visual cortex layer 2/3

Extracted molecule

total RNA

Extraction protocol

Mice aged P35 or older were anaesthetized with ketamine and xylazine (100mg*kg-1 and 10mg*kg-1, respectively), perfused transcardially with cold sucrose solution (in mM: NaCl, 83;
KCl, 2.5; MgSO4, 3.3; NaH2PO4, 1; NaHCO3, 26.2; D-glucose, 22; sucrose, 72; and CaCl2, 0.5, bubbled with 95% O2 and 5% CO2) and decapitated, and the visual cortex was cut into
300µm coronal or tangential sections in cold sucrose solution. Slices were incubated in sucrose solution in a submerged chamber at 34°C for 30min and then at room temperature (21°C) until
used for RNA harvest from patch-clamped cells.
RNA was harvested as described previously (Pfeffer et al., 2013). In brief, slices were mounted under a 40x 0.8 NA water lens and superfused with sucrose solution (same as in slice
preparation). Cells were identified using GFP or tdTomato fluorescence and visualized with IR-DIC. Cells were then patched with regular RNAse free patch pipettes (2-4 MOhm resistance) in
whole-cell mode using the voltage-clamp configuration. After seal establishment and break-in the cell was held at -70mV and the cell content including cell nucleus was harvested using
negative pressure and expelled in an Eppendorf tube and frozen at -70C. Intracellular solution was RNAse free: in mM: CsMeSO4, 115; NaCl, 4; HEPES, 10; Na3GTP, 0.3; MgATP, 4; EGTA,
0.3; BAPTA(4Cs), 10; adjusted to pH 7.4 with CsOH; mOsm 295. The procedures lasted approximately 2-3 minutes per cell (from targeting to tube freezing).
Single cell RNA-seq was carried out following the Cel-seq protocol (Hashimshony et al., 2016; Hashimshony et al., 2012). RT and second strand synthesis were performed using Superscript
III (Invitrogen) and second strand synthesis kit (Invitrogen), respectively, before IVT amplification (Maxiscript T7, Ambion). 96 samples (up to 94 cells and 2-8 control samples) were
handled in parallel to construct one sequencing library. After library fragmentation (Mg-Acetat + heat) and 3’ adaptor attachment, 11-15 cycles of PCR were used for library amplification (final
concentration > 1ng/microL, library peak fragment size ~350 nt). Libraries were sequenced using Illumina HiSeq 2500 and HiSeq 4000 at the UCSD genomics core (1st read 13 cycles for
the barcode, 2nd read 50 cycles for the 3’ end of the transcript).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

CP-D_47
SOM-PV_tpm5.txt
PV-VIP_tpm5.txt

Data processing

Illumina Casava 1.8 for basecalling
Reads were demultiplexed, trimmed, and filtered (prinseq, parameters: -lc_method dust –lc_threshold 15 –trim_left 7) before mapping (bowtie2, standard parameters; (Langmead and
Salzberg, 2012)) the reads to the mouse transcriptome (ENSEMBLE cDNA sequences or ab initio predicted genes), the ERCC transcriptome and mapped reads counted (htseq-count;
(Anders et al., 2014)) following the Cel-seq general pipeline.
Genome_build: Ensemble cDNA sequences or ab initio predicted genes; mouse ensemble release v76; GRCm38.p2
Supplementary_files_format_and_content: tab-delimited text files include tpm values for each Sample

Submission date

Dec 02, 2016

Last update date

May 15, 2019

Contact name

Carsten Pfeffer

E-mail(s)

cpfeffer10@gmail.com

Organization name

UCSD

Street address

9500 Gilman Drive