Nextflex chip seq barcodes

Embryo RNA‐Seq libraries were prepared using Nugen Ovation RNA‐Seq Systems for Model Organisms starting with 70–90 ng of total RNA. Seedling RNA‐Seq libraries were constructed from 5 μg of total RNA, using the library preparation protocol described by Kumar et al. (2012 ), with the exception of the RNA and mRNA isolations (polyA RNA was isolated from total RNA as described in the Supplementary Methods 2 of Kumar et al. (2012 ). The NEXTflex ChIP‐Seq Barcodes (BioScientific) were used as Illumina‐compatible adapters. Libraries were quantified using Quant‐iT PicoGreen dsDNA Reagent (Grand Island, NY) and a Nanodrop ND‐3300 instrument (Thermo Fisher Scientific) and sequenced on a HiSeq 4000 sequencer (Illumina). The sequencing was carried by the DNA Technologies and Expression Analysis Core at the UC Davis Genome Center.
Sequenced reads were demultiplexed, quality‐filtered, and reads corresponding to rRNA sequences were removed. The resulting filtered reads were mapped to Arabidopsis primary transcripts (TAIR10) using bowtie v0.12.7 with parameters ‐v 2 ‐5 10 ‐3 40 ‐m 1 –best ‐‐strata.
We used the EdgeR package (v3.10.5) to obtain normalized expression values using the Trimmed Mean of M‐values (TMM) method and to identify differentially expressed genes (DEGs) between the different genotypes (FDR < 0.05, Robinson et al., 2010 (link)).