title: GSM5583322: 8DAF-3 resequenced; Camelina sativa; RNA-Seq
library_construction_protocol: DAP-seq: Camelina sativa genomic DNA were extracted using urea buffer (7M urea, 350mM NaCl, 50mM Tris-HCl pH8, 20mM EDTA, 1% N-lauroyl sarcosine) and mixed with phenol:chloroform:isoamyl alcohol 25:24:1. The supernatants containing DNA were further precipitated using 3M NaOAc (pH 5.2) and isopropanol followed by 70% ethanol wash. The DNA pellet was resuspended in UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen) followed by RNase A (Roche) treatment and ethanol precipitation. RNA-seq: Total RNA from fresh seed was extracted using Spectrum Plant Total RNA Kit (Sigma-Aldrich) according to the manufacturer's protocol. The total RNA was prepared with three biological replicates, each with ~100 mg seeds. The quality of total RNA was determined by TapeStation4200 (Agilent), and cDNA library was generated with 1 μg of total RNA using TruSeq stranded mRNA (Illumina) DAP-seq: gDNA libraries were constructed following the protocol of Bartlett et al., 2017 with minor modifications. Extracted gDNA were fragmented to the size range between 200-400 bp using Diagenode's Bioruptor® 300 for 40 cycles with 30 seconds on/off at high energy. The fragmented DNA was further used for end repair and adapter ligation. To create modification free DNA, additional 11 cycles of PCR amplifications were performed using the adapter ligated libraries, followed by ethanol precipitation. Finally, the amplified gDNA libraries (ampDAP) were used for all protein-DNA interaction procedures. All the buffers and procedures for DNA-protein interaction were as published (Bartlett et al., 2017). except that the input gDNA library amount and the final step of library size selection. About 200 ng of ampDAP gDNA library were added as an input to mix with each pIX-HALO-TF protein. Finally, to perform double-size selection targeting 300-400 bp fragments, 0.7 volume of Agencourt AMPure XP beads (35 µl) to 1 volume of sample (50 µl) were mixed for 5 minutes and the bead was discarded to remove fragments with size larger than 400 bp. Next, the supernatant (85 µl) containing < 400 bp fragments were added to 0.2 volume of Agencourt AMPure XP beads (10 µl) and mixed for 15 minutes. The bound fragments were eluted from the beads by adding 18 ml UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen). The concentrations of eluted DNA were measured using the Qubit HS dsDNA assay kit, and approximate 5-20 ng/µl final concentrations were obtained. The fragment size and binding capacity to the flow cell were further examined on the agarose gel by six cycles of PCR using 2 µl of eluted ampDAP-seq library with Illumina P5 and P7 primers. Twelve libraries were pooled in one lane and sequenced by Illumina HiSeq 4000 SE50 at RTSF Genomics core at Michigan State University. RNA-seq : Total RNA from fresh seed was extracted using Spectrum Plant Total RNA Kit (Sigma-Aldrich) according to the manufacturer's protocol. The total RNA was prepared with three biological replicates, each with ~100 mg seeds. The quality of total RNA was determined by TapeStation4200 (Agilent), and cDNA library was generated with 1 μg of total RNA using TruSeq stranded mRNA (Illumina). The pooled libraries were sequenced with a paired-end read length of 150 bp by Illumina HiSeq 4000 at the Research Technology Support Facility Genomics Core at Michigan State University.