|
| Status |
Public on Oct 01, 2019 |
| Title |
Phf21b_ATAC_N2A_siPhf21b_Undf_Replicate1 |
| Sample type |
SRA |
| |
|
| Source name |
N2A_siPhf21b_Undf_Replicate1
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Neuro2a
replicate: Replicate1
developmental stage: Undifferentiated
|
| 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 |
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.
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 |
genomic DNA |
| Extraction protocol |
For ATAC seq the protocol from Buenrostro was followed.
Libraries for ATAC seq were prepared using Nextera DNA Sample Preparation Kit.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ATAC-seq in N2A
|
| Data processing |
For the ATAC-Seq samples, sequenced reads were aligned to mouse reference genome (mm9) using Bowtie v0.12.9 with the following parameters; [bowtie -S -p 10 -X 2000]. SAMTOOLS v0.1.19 was used to convert the SAM file into BAM format and to sort and index the BAM file. From the sorted BAM file, peak calling was performed using MACS2 v2.0.10 with BAMPE option. Generation of WIG files, and calculation of enrichment scores above input for each of the peaks were performed using QuasR.
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.
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| |
|
| 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.
|
| City |
Mainz |
| State/province |
Germany |
| ZIP/Postal code |
55112 |
| Country |
Germany |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE106999 |
Phf21b is a novel histone reader essential for epigenetic silencing of cell cycle genes during neurogenesis |
|
| Relations |
| BioSample |
SAMN08032750 |
| SRA |
SRX3399001 |
