|
| Status |
Public on Jun 27, 2019 |
| Title |
5_diff155_32_A8 |
| Sample type |
SRA |
| |
|
| Source name |
LUHMES-derived neurons
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: LUHMES-derived neurons
cell line name: OEC
differentiation: 155
developmental stage: day9
mecp2 expression level: 1.10
|
| Treatment protocol |
Cells were modified using CRISPR system for KO and infected with lentiviruses carrying MeCP2 for OE.
|
| Growth protocol |
LUHMES cells were cultured and differentiated according to published protocol (Shah et al., 2016).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from all generated cell lines at day 9 of differentiation using either an RNeasy Mini kit or an AllPrep DNA/RNA Mini kit (Qiagen). Genomic DNA contamination was removed with a DNA-free kit (Ambion) and remaining DNA-free RNA was tested for purity using PCR for GAPDH genomic locus. Total RNA was tested on 2100 Bioanalyzer (Agilent Technologies) to ensure a RIN quality higher than 9 and quantified using Nanodrop.
Equal amounts of total RNA (1 µg to 2.5 µg) were taken forward for library preparation and ERCC RNA Spike-in control mixes (Ambion) were added according to manufacturer guide. Ribosomal RNA was depleted using the Ribo-Zero Gold rRNA Removal module (Epicentre, Illumina) and isolated mRNA was tested for purity on 2100 Bioanalyzer (Agilent Technologies). mRNA was quantified with Qubit and equal amounts used for cDNA synthesis and 3’ terminal tagging using ScriptSeq v2 RNA-seq library preparation kit (Epicentre, Illumina). The library was PCR amplified to add adaptors and barcodes.
ScriptSeq v2 RNA-seq library preparation kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Sequenced reads were trimmed for adaptor sequence, and filtered for low-complexity or low-quality sequence using Trimmomatic v0.33 with arguments -threads 10 input.1.fastq.gz input.2.fastq.gz output.1.trimmed.fastq.gz output.1U.trimmed.fastq.gz output.2.trimmed.fastq.gz output.2U.trimmed.fastq.gz ILLUMINACLIP:TruSeq2-PE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:30 MINLEN:30
Trimmed and high quality reads were aligned using STAR v2.4.2a on index created using hg19 genome assembly and Ensembl 74 annotation. Software parameters are as follows STAR --runThreadN 10 --runMode alignReads --genomeDir hg19 --genomeLoad LoadAndKeep --readFilesCommand zcat --outFilterIntronMoti fs RemoveNoncanonicalUnannotated --outSAMtype BAM SortedByCoordinate --limitBAMsortRAM 80000000000 --outFileNamePrefix output.STAR. --readFilesIn output.1.trimmed.fastq.gz output.2.trimmed.fastq.gz
Normalized bigwig tracks were generated using Bedtools genomeCoverageBed program with parameters genomeCoverageBed -ibam input.bam -split -bg -g hg19.fa.fai -scale no_mapped_reads > output.bg and then bedgraph files were converted to bigwig files using UCSC tools bedGraphToBigWig utility
SubRead v1.5.0 package was use to assign read pairs to genes and generate count tables. The following parameters were used: featureCounts -T 10 -p -s 1 –primary -t exon -g gene_id -a annotation.gtf -o counts.tsv input.bam
Sequenced reads were quasi-mapped and gene expression was quantified using sailfish v0.10.0 using Gencode v19 annotation as index. The following parameters were used: sailfish quant -i gencode.v19.index -o outdir -p 12 -1 input.1.fastq.gz -2 input.2.fastq.gz –enforceLibCompat -g gencode.v19.index.genes –numBootstraps 20 –biasCorrect -l IU
Genome_build: hg19
Supplementary_files_format_and_content: bigWig
|
| |
|
| Submission date |
Feb 15, 2019 |
| Last update date |
Jun 27, 2019 |
| Contact name |
Shaun Michael Webb |
| E-mail(s) |
shaun.webb@ed.ac.uk
|
| Organization name |
University of Edinburgh
|
| Department |
Wellcome Trust Centre for Cell Biology
|
| Lab |
Bioinformatics Core Facility
|
| Street address |
2.21 Swann Building, Kings Buildings
|
| City |
Edinburgh |
| ZIP/Postal code |
EH9 3JR |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE125660 |
Study of MeCP2 in LUHMES-derived neurons |
| GSE126640 |
Investigation of transcriptional changes in cells expressing different levels of MeCP2 |
|
| Relations |
| BioSample |
SAMN10954167 |
| SRA |
SRX5383229 |
