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Sample GSM4588497

Query DataSets for GSM4588497

Status

Public on Dec 31, 2021

Title

Co.IP.Anti.ORRM1.A

Sample type

SRA

Source name

Chloroplasts

Organism

Zea mays

Characteristics

tissue: Chloroplasts from 14-21 day old seedlings
cultivar: Sugar Buns
antibody: Anti-ORRM1
fraction: Bound beads
age: 14-21 day old seedlings
tissue: leaf

Treatment protocol

Leaf tissue was homogenized in a blender and intact chloroplasts isolated using a continuous Percol gradient.

Growth protocol

Zea mays var. Sugar Buns (Johnny’s Selected Seeds, Fair- field, ME, USA) seeds were planted and grown for 14-21 days in a greenhouse.

Extracted molecule

total RNA

Extraction protocol

Pre-cleared chloroplast supernatant was placed in the chilled antibody-beads tube, mixed gently by pipetting, and incubated for 20 min at 4°C.
Library construction was performed according to the Illumina TruSeq® Stranded mRNA Sample Preparation Guide with modification. The input quantity for total RNA was 50 ng and chemically fragmented for three minutes. First strand synthesis used random primers and reverse transcriptase was used to make cDNA. After second strand synthesis the ds cDNA was cleaned using AMPure XP beads and the cDNA was end repaired and then the 3’ ends were adenylated. Illumina barcoded adapters were ligated on the ends and the adapter ligated fragments were enriched by nine cycles of PCR. The resulting libraries were validated by qPCR and sized by Agilent Bioanalyzer DNA high sensitivity chip. The concentrations for the libraries were normalized and then multiplexed together. The multiplexed libraries were sequenced on the Miseq using version 3 single end 150 cycles chemistry. The version of Miseq control software was MCS v: 2.6.2.1 with real time analysis software, RTA 1.18.54.0.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina MiSeq

Data processing

Phred built into Trimmomatic was used for basecalls.
Bad quality reads and adapter sequences were trimmed from the MiSeq libraries using Trimmomatic (Bolger et al., 2014). Next the trimmed datasets were aligned to the maize reference genome B73_RefGen_v4 (Jiao et al., 2017) using bowtie2, tophat2 and cufflink (Langmead and Salzberg, 2012, Trapnell et al., 2012). Resulting files were converted into SAM and BAM format using SAMtools (Li et al., 2009). Performed using a custon script. #java -jar /usr/local/bin/trimmomatic-0.35.jar SE -phred33 mR134-L1-P1-CGATGT CPXRNA.fq ILLUMINACLIP:/usr/local/bin/adapters/TruSeq3-SE.fa:2:30:10 LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15 MINLEN:36; bowtie2-build [infile fasta] Zea_mays_4_cp; bowtie2 -x Zea_mays_4_cp -U CPXRNA.fq -S CPX.sam; samtools view -S -b CPXRNA.sam > CPXRNA.bam; samtools sort -o CPXRNA.bam CPXRNA.sorted.bam; samtools index CPXRNA.sorted.bam; featureCounts -T 5 -t exon -g gene_id -a ZM_v4.43.gtf -O -o Result.txt CPXRNA sorted.bam#
RPKM was calculated by taking the total number of reads assigned to unique gene features from Zea_mays.B73_RefGen_v4.46.chromosome.Pt.gff3 from Ensembl Genomes and then using the equation [feature reads / (gene length (kb)/ total mapped reads/1,000,000)]
Reads from feature count were exported into a single data sheet using featureCounts.
Redundant reads from FeatureCount that map twice to the inverted repeat were removed to only count single, unique gene features
Genome_build: B73_RefGen_v4
Supplementary_files_format_and_content: BAM files used for genome alignmnets
Supplementary_files_format_and_content: Table including all raw reads and processed RPKM and fold change values

Submission date

Jun 03, 2020

Last update date

Dec 31, 2021

Contact name

Michael Lloyd Hayes

E-mail(s)

[email protected]

Organization name

California State University Los Angeles

Department

Chemistry and Biochemistry