NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5683928 Query DataSets for GSM5683928
Status Public on Jan 23, 2022
Title NGN2_RNA_seq_CD11_AG_sample2
Sample type SRA
 
Source name 81566
Organism Homo sapiens
Characteristics cell type: Glutamatergic, NGN2-induced, day-20 neuron
rutgers university cell and dna repository rucdr-nimh stem cell center index id: 05C46807
disease status: case
gender: Female
age: 58
Extracted molecule total RNA
Extraction protocol For RNA-Seq, at the day of harvesting the culture media was removed by aspiration and the cells were washed twice using 1 ml PBS each with aspiration. Subsequently, 600 µl of RLT Plus reagent was directly added into each well to homogenise cells in situ. Qiagen RNEasy plus kit was used for the extraction of total RNA according to the manufacturer’s protocol. Extracted RNAs were eluted into 50 µl of RNA-free water and the concentration was determined by nanodrop. Approximately 5 µg total RNA was recovered from each well.
Total RNAs isolated by using MirVana kit (Thermofisher) were used in RNA-seq. After QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Subsequently, terminal repair and sequencing adaptor ligation were applied to the templates. The double-stranded cDNA library is completed through size selection and PCR enrichment.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description NGN2_RNA_seq_CD11_AG_sample2 [CD0000011]
Data processing All raw sequence reads generated by Illumina HiSeq 2000 had been demultiplexed at the University of Minnesota Genomics Center and provided as 2×75 bp paired-end fastq files (targeting 60 M reads per sample).
Adapter remnants, low-quality reads, and low QSEQ short sequences near either end of reads were processed by Trimmomatic (ILLUMINACLIP:NexteraPE-PE.fa:2:30:7, SLIDINGWINDOW:3:18, MINLENGTH:26).
The processed sequences were separated into paired-end and single-end fastq files per sample and only paired-end reads were retained for subsequent mapping. The fastq files were individually mapped against the human genome reference file including decoy sequences (GRCh38p7.13/hg38, 1000 Genome Project) using bowtie2 (-x 2000, -mm --qc-filter –met 1 –sensitive –no-mixed -t) and subsequently merged and sorted as BAM-formatted files using samtools, with only uniquely mapped reads (MAPQ > 30) retained. Picard tools MarkDuplicate was then used to remove all PCR and optical duplicated reads from the BAM file.
To further eliminate allelic bias towards reference alleles during the aligning step, we performed WASP calibration on the generated raw BAM files 55. Briefly, we first called the VCF file profiles on all SNP sites that were not reference alleles of all 20 samples individually using GATK HaplotypeCaller, and subsequently collapsed the individual VCF files into one summary VCF file containing all non-reference sites of all 20 individuals. Subsequently, this SNP list was used as the basis of WASP calibration and re-alignment, and a new WASP-calibrated new BAM file set was collected as the final output for the following peak calling and ASoC SNP call 55.
In order to increase sample size and sensitivity for peak detection, the BAM files of the processed reads of each sample were first sub-sampled to 30 M paired-end reads per sample (the smallest sample size) and the 20 sub-sampled BAM files were merged as the source file for peak calling. MACS2 26 was used to generate peak files (narrowPeak format) with recommended settings at FDR = 0.05 (-f BAMPE, --nomodel, --call-summits --keep-dup-all -B). Peaks that fell within the ENCODE blacklisted regions were removed. Also, we removed peaks falling within chromosomes X and Y, and the mitochondrial genome regions.
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: .narrowPeak, MACS2 peak file
 
Submission date Nov 09, 2021
Last update date Jan 23, 2022
Contact name Jubao Duan
E-mail(s) jduan69@gmail.com
Phone (224) 364-7564
Organization name NorthShore University HealthSystem
Department Center for Psychiatric Genetics
Lab Unit of Functional Genomics in Psychiatry
Street address 1001 University Place
City Evanston
State/province IL
ZIP/Postal code 60201
Country USA
 
Platform ID GPL11154
Series (1)
GSE188491 Chromatin accessibility mapping at schizophrenia GWAS risk loci reveals compound genetic effects impairing neurodevelopment and synaptic function in human neurons
Relations
BioSample SAMN22959287
SRA SRX13025757

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap