title: GSM2306486: dim-5_polyA-mRNAseq; Neurospora crassa; RNA-Seq
library_construction_protocol: protocol for mRNA isolation and library preparation: Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were harvested and added to a tube containing ~300uL glass beads (260-300 micrometer, acid washed [Sigma Aldrich] and resuspended in sdH2O), 350uL NETS buffer (10mM Tris pH 7.5, 300mM NaCl, 1mM EDTA, 0.2% SDS) and 350uL phenol:chloroform:isoamyl alcohol (25:24:1) and lysed by a bead beater for 10 minutes at room temperature. Samples were cleared by centrifugation, and the aqueous (top) phase was added to 650uL cold 100% EtOH.  Samples were precipitated at -20oC, pelleted by centrifugation, washed with 70% EtOH, and resuspended in DEPC-treated dH2O.RNA was treated with DNase (DNA-free™ DNA Removal Kit, Thermo Fisher Scientific), cleaned (Agencourt® RNAclean XP® beads, Beckman Coulter), and RNA-seq libraries were prepared (KAPA Stranded mRNA-seq kit, KAPA Biosystems) protocol for histone modification ChIPseq: Cultures were grown in Vogels medium and 1.5% sucrose (standard minimal medium), shaking for 16 hours at 32oC. Mycelia were washed in PBS prior to crosslinking. Mycelia were cross-linked for 10 min in 0.5% formaldehyde for histone modification ChIP.  Tissue added to ChIP lysis buffer (50mM Hepes pH 7.5, 90mM NaCl, 1mM EDTA, 1% triton + proteinase inhibitors) and was disrupted by sonication for thirty pulses before chromatin was sheared using a Bioruptor (Diagenode) for 15 min with a cycle of 30 sec on followed by 30 sec off, at high power. Lysates were cleared by centrifugation, 1/20th Input was saved, and histone modification antibody was added (typically 1uL) and samples were rotated overnight at 4oC. The next day, equilibrated Protein A or Protein G slurry (agarose or magnetic beads) was added to bind the antibody, incubated for 3 hours at 4oC, and washed twice in cold ChIP Lysis buffer, once with ChIP Lysis buffer + 0.5M NaCl, once with LiCl was buffer (10mM Tris pH 8.0, 250mM LiCl, 1mM EDTA, 0.5% NP40), and DNA/protein was eluted with TES buffer (50mM Tris pH 8.0, 10mM EDTA, 1% SDS) and incubated at 65oC.  Samples (IP and input) were decrosslinked at 65oC overnight.  The next day, samples were proteinase K treated for 2 hours at 50oC, and DNA was purified using the QIAquick PCR purification kit  and eluted in 30 μl of water.  Approximately 10 ng of DNA was used to generate ChIP-Seq libraries.  Each library was prepared using the Illumina Tru-Seq kits A and B (Illumina, IP-202-1012 and IP-202-1024) according to the manufacturer’s instructions.  “Invisible” fragments between 250-400 bp were excised and purified using the MinElute gel extraction kit (Qiagen, 28606).  Final libraries were PCR-amplified using one cycle at 98 °C for 30 sec, 8 cycles at 98 °C for 10 sec, 60 °C for 30 sec and 72 °C for 30 sec and a final extension at 72 °C for 5 min. Indexed paired-end HiC libraries were pooled and sequenced either on an Illumina HiSeq 2500 as a 50nt paired end sequencing run, or a NextSeq500 as either 37nt or 75nt paired-end sequencing run at the University of Oregon Genomics Core Facility. ChIP-seq or RNA-seq samples were sequenced on a NextSeq500 as a single-end 75nt run.
attributes.tag: GEO Accession
attributes.value: GSM2306486