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Sample GSM3057022

Query DataSets for GSM3057022

Status

Public on Mar 30, 2019

Title

T21_dnt_RNAseq_1

Sample type

SRA

Source name

dorsal neural tube (dnt)

Organism

Petromyzon marinus

Characteristics

Stage: T21
tissue: dorsal neural tube (dnt)
number of individuals per sample: 35

Extracted molecule

polyA RNA

Extraction protocol

T18, T20 and T21 dorsal neural tubes including premigratory, migrating and/or post-migratory neural crest cells were dissected from the head using an eye-lash knife. T20 and T23 heads were dissected using forceps.
For RNA-seq, tissue was lysed in the Ambion RNaqueous Total RNA Isolation kit lysis buffer (AM1931), set on ice for 15 minutes with occasional vortexing, flash frozen in liquid nitrogen and stored at -80°C. RNA was extracted using the Ambion RNAqueous Micro Total RNA isolation kit and assessed using the Agilent Bioanalyser.
For ATAC-seq, groups of dissected tissue were collected into L-15 medium (Lifetech) with 10% fetal bovine serum at 19°C. Tissue was first dissociated in dispase (1.5 mg/ml in DMEM; 10mM Hepes, pH 7.5), followed by the addition of an equal volume of trypsin (0.05% Trypsin; 0.53mM EDTA in HBSS) at room temperature for a total of up to 15 minutes. Dissociated cells were passed over a 40 μm cell strainer into Hanks’ solution (1xHBSS; 10mMHepes; 0.25%BSA) and centrifuged at 500 x g for 7 minutes at room temperature. The supernatant was removed and fresh Hank’s solution applied. 50 000 cells were counted out and centrifuged for 5 minutes at 500 × g at 4°C and washed with cold 2/3 phosphate buffered saline (PBS) by centrifugation for 5 minutes at 500 × g, 4°C.  
RNA-seq libraries were prepared from 100 ng RNA per sample using the NEBNExt Ultra Directional RNA Library Prep Kit for Illumina (E7420) in combination with the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext High-Fidelity 2X PCR Master Mix (M0451S). Libraries were indexed and enriched by 15 cycles of amplification. Library preparation was assessed using the Agilent TapeStation and libraries quantified by Qubit. The concentration of library pools was assessed with the KAPA Library Quantification Kit (KK4835).
For ATAC-seq, dissociated cells were lysed (10mM Tris-HCl, pH7.4; 10mM NaCl; 3mM MgCl2; 0.1% Igepal) and tagmented using the Illumina Nextera kit (FC-121-1030) for 30 minutes at 37°C as previously described (Buenrostro et al. 2015 Curr Protoc Mol Biol 109, 21 29 21-29, doi:10.1002/0471142727.mb2129s109), with the addition of a tagmentation-stop step by the addition of EDTA to final concentration of 50 nM and incubation at 50°C for 30 minutes. Tagmented DNA was amplified using the NEB Q5 High-Fidelity 2X Master Mix (M0492S) for 14 cycles. Tagmentation efficiency was assessed using Agilent TapeStation and libraries quantified by Qubit. The concentration of ATAC library pools was assessed with the KAPA Library Quantification Kit (KK4835).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

RNAseq_transcriptome.gtf
RNAseq_Featurecounts.txt
RNAseq_DESeq2_normalised_counts.txt

Data processing

RNA-seq: Reads were mapped to the sea lamprey germline genome assembly using STAR (v2.4.2 )63 (STAR --genomeDir $GENOME --readFilesIn $R1.fastq $R2.fastq --runThreadN 4 --outFileNamePrefix $PREFIX --readFilesCommand zcat --outSAMstrandField intronMotif --alignEndsType EndToEnd --outReadsUnmapped Fastx --outSAMtype BAM SortedByCoordinate). Separate transcriptomes for dorsal neural tube sample datasets or head and embryo sample datasets were assembled de novo with Cufflinks followed by Cuffmerge using default parameters to make a consensus transcriptome from all the datasets. Read counts for dorsal neural tube datasets were obtained with Subread featureCounts (v1.4.6-p4) using the Cuffmerge consensus transcriptome in SAF format as a reference (featureCounts -p -B -M -F SAF -s 2 -T 4 -a $SAF -o $OUT $IN.bam). Differential expression analysis was performed on the dorsal neural tube read count datasets using DESeq2 (v.1.8.2)
ATAC-seq: Reads were mapped to the sea lamprey germline genome assembly using Bowtie2 (bowtie2 --phred33 -p 4 -X 2000 --very-sensitive -x $GENOME -1 $R1.fastq -2 $R2.fastq -S $OUT.sam). Duplicates were removed with Picard (v1.83) MarkDuplicates and the distribution of fragment sizes assessed with Picard (v1.83) CollectInsertSizeMetrics. Replicate bam files for each developmental stage were merged with SAMtools and filtered with BamTools to remove unpaired reads and reads mapped to the mitochondrial chromosome. Filtered bam files were down-sampled to match the file with the lowest number of reads using Picard (v1.83) DownsampleSam. Down-sampled bam files were sorted by name using SAMtools and paired-end bed files were obtained using bedtools(v.2.15.0) bamtobed bedpe. Reads were extended to a read length of 100bp. Peak-calling was performed using MACS2 (macs2 callpeak -t $IN.bed –f BED –name $IN.macs2 --outdir $OUT --shiftsize=100 --nomodel --slocal 1000). Output peak files (.xls) for each developmental stage were converted to bed format and merged with bedtools merge to create one consensus peak set.
Genome_build: Petromyzon marinus germline genome
Supplementary_files_format_and_content: RNAseq_transcriptome.gtf: Separate transcriptomes for dorsal neural tube sample datasets or head and embryo sample datasets were assembled de novo with Cufflinks followed by Cuffmerge using default parameters to make a consensus transcriptome from all the datasets.
Supplementary_files_format_and_content: RNAseq_Featurecounts.txt: a matrix of readcounts for the dorsal neural tube RNA-seq datasets generated with featureCounts
Supplementary_files_format_and_content: RNAseq_DESeq2_normalised_counts.txt: a matrix of normlaised readcounts for the dorsal neural tube RNA-seq datasets generated with DESeq2
Supplementary_files_format_and_content: Pm_ATAC_consensus_peaks.bb: Output peak files from MACS2 (.xls) for each developmental stage were converted to bed format and merged with bedtools merge to create one consensus peak set. Bed files were converted to bigBed with KentUtilities's bedToBigBed
Supplementary_files_format_and_content: Lp_ATAC_consensus_peaks.bb: Output peak files from MACS2 (.xls) for each developmental stage were converted to bed format and merged with bedtools merge to create one consensus peak set. Bed files were converted to bigBed with KentUtilities's bedToBigBed

Submission date

Mar 20, 2018

Last update date

Mar 30, 2019

Contact name

Tatjana Sauka-Spengler

E-mail(s)

tatjana.sauka-spengler@imm.ox.ac.uk

Organization name

MRC Weatherall Institute of Molecular Medicine

Department

University of Oxford