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Sample GSM2859275

Query DataSets for GSM2859275

Status

Public on Oct 01, 2019

Title

Phf21b_RNA_IUE_shPhf21b_Replicate1

Sample type

SRA

Source name

IUE_shPhf21b_Replicate1

Organism

Mus musculus

Characteristics

replicate: Replicate1
developmental stage: E17.5 embryo
tissue: cortex
strain: C57BL6/J
genotype/variation: Wild type

Treatment protocol

(1) For ES cell ChIP and RNA seq: Transgenic A2lox ES cells harboring the murine Phf21b fused to an N-terminal HA-tag under the control of a doxycycline-inducible promoter were generated according to Iacovino et al (2011). Ectopic induction of Phf21b was achieved with 500 ng/ml doxycycline for 24 hours.2) ChIP-seq, ATAC seq and RNA seq from N2a day2 neuronal differentiation in N2A cells was induced by adding 20 μM retinoic acid in DMEM supplemented with 2% FBS, 1% Glutamine and 1% NEAA. (3) For all siRNA-mediated knockdown experiments, cells were seeded and pre-depleted for two days by transfecting with ON-TARGETplus SMARTpool siRNAs (i.e., a mixture of 4 siRNAs provided as a single reagent from Dharmacon) every second day. For siRNA transfections, Lipofectamine RNAiMax was used according to the manufacturer's instructions. For N2a differentiation pre-depleted cells were differentiated by adding retinoic acid along with siRNAs for two days in N2a differentiation media.

Growth protocol

For in vivo RNA seq, we carried out IUEs of progenitor cells at E13.5 mouse cortex and FACS sorted the electroporated cells 4 days after IUE.
Murine Neuro2a cells (N2a cells) were cultured at 37°C in 7% CO2 and 88% relative humidity in 10 ml of DMEM supplemented with 10% fetal calf serum, 1× NEAA and 2 mM L-glutamine. Neuronal differentiation in N2A cells was induced by adding 20 μM retinoic acid in DMEM supplemented with 2% FBS, 1% Glutamine and 1% NEAA. For ES cell experiments, ES159 cells on feeders were splitted every 2 days onto tissue culture dishes coated with 0.2% gelatin, and the media was changed daily. All ES cell experiments were performed at feeder-free five stage. In vitro neuronal differentiation was performed as described by Bibel et al (2004, 2007) where ES cells were directly plated on PORN/Laminin-coated tissue culture dishes and cultivated for 2 days in N2 media followed by complete media for induced neuron (iTN) formation.

Extracted molecule

total RNA

Extraction protocol

RNA samples for in vivo RNA seq from cortex was FACS sorted and isolated by RNeasy Micro Kit
RNA libraries for the in-vivo cortex RNA seq Ovation® SoLo RNA-Seq System was used.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

RNA-seq in vivo cortex
Normalized_counts_IUE.txt
IUE_shPhf21b_Replicate1

Data processing

For the low-input in vivo RNA-Seq samples, sequenced reads were aligned to the mouse reference genome (mm9) with UCSC’s annotations using TopHat v2.0.8. Unique molecular identifiers (UMI) were added to the sorted bam file using AddUMIsToBam function in bamtools and then unique read count was calculated using picard-2.9.0 MarkDuplicates function with following option: [REMOVE_DUPLICATES=true BARCODE_TAG=RX ASSUME_SORTED=true]. SAMTOOLS v0.1.19 was used to convert the BAM output to SAM format and to sort the BAM file. From the aligned BAM files, the read counts per gene were calculated using the HTSeq program v0.5.4p1. Read counts from HTSeq program was normalized using DESeq program.
Genome_build: mm9
Supplementary_files_format_and_content: For the RNA-Seq samples, tab-delimited text files include DESeq normalized read counts for each Sample. For the ChIP-Seq and ATAC-Seq samples, wig files for UCSC genome browser track were provided.

Submission date

Nov 16, 2017

Last update date

Oct 01, 2019

Contact name

Hyobin Jeong

E-mail(s)

h.jeong@imb-mainz.de

Organization name

IMB

Street address

Richard shirrmann Str 12.