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Sample GSM954621

Query DataSets for GSM954621

Status

Public on Dec 13, 2012

Title

H2AZ_WT_3_RNA

Sample type

SRA

Source name

H2AZ-WT Rosettes 4 weeks

Organism

Arabidopsis thaliana

Characteristics

genotype/variation: h2a.z-related wild type (F2 sibling)
tissue: rosettes
age: 4 weeks

Growth protocol

Arabidopsis plants were grown according to one of the following methods: 1) For bisulfite sequencing of seedling tissues, seeds were planted on 1x Murashige and Skoog Media with micronutrients and 1.5% Sucrose (Caisson Laboratories) and grown under 16h light/ 8h dark for 14 days in a growth chamber. 2) For bisulfite sequencing of rosette and cauline leaf tissue, seeds were planted on soil and grown in greenhouse conditions with LD 16h light / 8h dark. 3) For phenotype analysis of the h2a.z mutant, seeds were planted on soil and grown in greenhouse conditions with either 16h light / 8h dark (LD) or 8h light/ 16h dark (SD).

Extracted molecule

polyA RNA

Extraction protocol

For Bisulfite-Sequencing, approximately 100-500 ng genomic DNA was isolated from either seedling, rosette or cauline leaf tissues. Seedling tissue was obtained from 14 days post germination seedlings grown on Murishige and Skoog media in LD (16h light/ 8h dark). Mature rosette leaves and mature cauline leaves were obtained from 4 week post germination mature plants grown on soil in LD (16h light/ 8h dark). In general, multiple biological replicates were generated for each mutant and WT line; a complete list of all generated libraries is available in Table S4. WT datasets for each mutant were generated from plants derived from recent relatives of the relevant mutant. Bisulfite conversion and Illumina library construction and sequencing were performed as described previously. We used single ends (SE) Illumina sequencing for bisulfite sequencing on the GAII and HiSeq platforms and sequence alignments were performed using Bowtie and the TAIR8 Genome Annotation (http://www.arabidopsis.org/) as described previously. The average percent methylation plots were generated as described previously. For RNA -Sequencing, approximately 30 ug total RNA was isolated from 4 week post germination mature rosette leaves using the RNEasy Plant Extraction Kit (Qiagen) with the optional on-column DNAse treatment. mRNA was purified from total RNA by two treatments of poly-A enrichment using the Oligotex kit (Qiagen #72022), followed by a rRNA removal step using the RiboMinus Plant Kit for RNA-sequencing (Invitrogen #A1083702). Illumina library construction and RNA-sequencing were performed as described in [34]. We used single ends (SE) Illumina sequencing for RNA-sequencing on the GAII platform and sequence alignments were performed using Bowtie and the TAIR8 Genome Annotation and cDNA Annotation. For ChAP-Sequencing, H2A.Z-containing nucleosomes were chromatin affinity purified (ChAP) from 4 week post germination Arabidopsis roots of our H2A.Z-BLRP transgenic lines grown in LD conditions as in [16]. Illumina libraries were constructed for IP and input DNA samples and sequenced generating SE 50 bp reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina Genome Analyzer II

Data processing

For Bisulfite Sequencing, we used Perl scripts to convert all the Cs in the reads (and in the scaffolds) to Ts, and aligned the converted reads to the converted reference scaffold, allowing up to two mismatches per read. 76 base reads were divided into the first 45 and the last 31 bases. 50 base reads were processed as single reads. Each half of the read was aligned independently using bowtie, allowing up to two mismatches. The coordinates of the two halves were subsequently correlated; the second half was discarded if it did not match the first. Single_C: We used Perl scripts to recover the original sequence of each mapped read and, for each C (on either strand), count the number of times it was sequenced as a C or a T. 50bp_window: We used a Perl script to calculate fractional methylation (#C/(#C+#T)) within a 50 bp sliding window for each sequence context (CG, CHG, CHH).
For ChAP-Sequencing, nucleosomal midpoints were estimated based on an average 150-bp nucleosome length by adding 75bp to the start position of each read. Differences between IP and input over each single-base window were generated to give an overall genome-wide map of H2A.Z-enrichment. Genes were aligned at either their 5' or 3' ends, and average H2A.Z-enrichment values were calculated over each 50bp window between 3kb up and downstream of the point of alignment. Sequencing was carried out on the HiSeq platform and sequence alignments were performed using Bowtie and the TAIR8 Genome Annotation
Genome_build: TAIR8
Supplementary_files_format_and_content: For Bisulfite Sequencing gff files, they contain fractional methylation data either for individual cytosines (single-c) or in 50 bp windows. For ChAP-Sequencing gff files, they contain data either for individual basepairs (w1) or in 50 bp windows (w50) for the H2A.Z enrichment data (mutant - WT normalized read counts). For all three Sequence dataset types, including RNA-Sequencing, the raw reads are presented in FASTQ format.

Submission date

Jul 02, 2012

Last update date

May 15, 2019

Contact name

Toshiro Nishimura

E-mail(s)

tnish@berkeley.edu

Phone

5106429550

Organization name

University of California at Berkeley