ABSTRACT: This BioStudy presents the results of peak-calling for H3K27me3-marking (the PRC2 mark), based on ChIP-Seq data obtained using anti-H3K27me3 (the PRC2 mark) and anti-H3 (as control).
Shoot and root of seedlings at 3 days after germination induction (dag), when seedlings are still heterotrophous and rely on their carbon reserves from the seeds, were compared with shoot and root of seedlings at 7 dag, when potentially seedlings have become autotrophous and capable of photosynthesis.
Two conditions were considered : autotrophic and heterotrophic. In autotrophy, plants were grown on half MS agar plates; in heterotrophy, plants were grown on half MS + sucrose + DCMU, the latter blocking the transition of the seedlings from heterotrophy to autotrophy.
- Growth conditions:
* autotrophic: half Murashige & Skoog (MS) medium including vitamins, MES and 0.8% (w/v) plant agar. The seeds were stratified over 2 nights (ca 65 hrs) and placed in long-day conditions (110-120 µmol m-2 s-1, 16hrs light/8 hrs dark, 22 °C during light/20 °C during dark; light source: OSRAM FQ 54W/840 HO CONSTANT LUMILUX Cool White).
* heterotrophic: half Murashige & Skoog (MS) medium including vitamins, MES and 0.8% (w/v) plant agar + 1% sucrose + 10µM DCMU. The seeds were stratified over 2 nights (ca 65 hrs) and placed in long-day conditions (110-120 µmol m-2 s-1, 16hrs light/8 hrs dark, 22 °C during light/20 °C during dark; light source: OSRAM FQ 54W/840 HO CONSTANT LUMILUX Cool White).
- ChIP-Seq:
Chromatin immunoprecipitation was carried out using previously described protocol (Mozgová et al. 2015). In brief plant material was crosslinked using 1% formaldehyde for 10 min under vacuum and crosslinking was terminated using 0.125 M glycine for 5 min. Sheared chromatin (10 cycles, 30 s ON/ 30 s OFF using a Bioruptor® (Diagenode)) extracted from 100 mg material was equally divided between input and samples immunoprecipitated with anti-histone H3 (Merck, cat. no. 07-690); anti-H3K27me3 (Merck, cat. no. 07-449). Immunocomplexes were collected using Dynabeads® Protein A for Immunoprecipitation (Thermo, cat. no. 10001D) and washed 2 x 5 min with low-salt buffer (containing 150 mM NaCl), 2 x 5 min with high-salt buffer (containing 500 mM NaCl) and 1 x 5 min with LiCl-containing buffer. Recovered DNA was extracted using phenol-chloroform and precipitated by ethanol with addition of GlycoBlue™ Coprecipitant (Thermo, cat. no. AM9515).
Immunoprecipitated DNA was purified using iPure kit v2 (Diagenode, cat. no. C03010015). Anti-H3 and anti-H3K27me3 immunoprecipitated was quantified using Qubit® dsDNA HS Assay (Thermo, cat. no. Q32854) and 3 ng of DNA was used for Illumina sequencing library preparation using NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® NEB, cat. no. E7645S) according to manufacturer´s instructions. Libraries were pooled into two pools of 24 samples (expected output min 20 Mio reads per sample) and sequenced on the Illumina HiSeq Hi-Outputv4 platform in 125 bp paired-end (PE) mode.
- Peak-calling:
SICER was used with the following parameters: Redundancy threshold 25; Window size 200; Gap 600; Effective genome size 0.998444; FDR 0.05; Fragment size 150; Control: H3 enriched sample (pool of replicates); Sample: H3K27 enriched sample (poole of replicates). To clean raw reads data, TrimGalore v0.6.2 was used, with parameters: Paired; Trim N; Phred33; Stringency 6; Quality 20; Minimum length 20; Clip 10 bp from 3’ and 5bp from 5’. Filtered ChIp-Seq reads were mapped to TAIR10 Arabidopsis thaliana genome using Bowtie2 v2.2.9 enabling the option --no-mixed. The SAM file was processed (mapping quality filter, with a MAPQ threshold of 25, conversion to BAM format, sorting and indexing) using SAM tools v1.9. Following Spearman correlation analyses of the two biological replicates, reads from the two replicates were pooled in the final analyses. BedTools v2.26.0 was used to parse the BAM format to BED format.
