GSM2410966: nascent_RNA seq_A7_replicate2; Drosophila melanogaster; RNA-Seq
RNA-SEQ: Nascent-RNA sequencing was done using a urea-based method similar to NET-seq (CHURCHMAN AND WEISSMAN 2011; CHURCHMAN AND WEISSMAN 2012), and essentially as reported elsewhere (ALEKSEYENKO et al. 2015).  In short, 1x107 S2 cells were collected by centrifugation at 300 x g at 4°C, and homogenized in CKS buffer + SUPERase•In RNase inhibitor (Ambion AM2696) + ProteaseArrest (G-Biosciences 786-108) with 3 strokes through a 25G needle.  Nuclei were collected by centrifugation and resuspended in CF buffer + RNasin.  NUN buffer was added and samples were vortexed ~30sec until a wispy, filamentous precipitate was apparent.  This precipitate was spun down and washed 3 times with NUN buffer.  Samples were then treated with proteinaseK in CF buffer + 0.5% SDS at 55°C for 30 min.  Samples were then passed through a 25G needle 5 times to disrupt the chromatin, and incubated an additional 30 min at 55°C.  Samples were extracted twice with 25:24:1 phenol:chloroform:isoamyl alcohol, once with 24:1 chloroform:isoamyl alcohol, and ethanol precipitated overnight in the presence of glycogen (Ambion AM9510).  Resulting nucleic acids were treated with RNase-free TURBO DNase (Ambion AM2238) for 30 min at 37°C, with proteinaseK +SDS for an additional 5min at 37°C, and then extracted once each with 25:24:1 phenol:chloroform:isoamyl alcohol and 24:1 chloroform:isoamyl alcohol.  Nascent-RNA was ethanol precipitated overnight except without the addition of glycogen.  Illumina sequencing libraries were constructed using the NEBNext Ultra Directional RNA Library kit (NEB 7420). ChIP-SEQ: Using a protocol essentially described elsewhere (ALEKSEYENKO et al. 2008), 0.1 grams of embryos were collected and disrupted using a motorized pestle.  Formaldehyde was added to 1% final concentration and incubated for 15min at room temperature.  Following quenching of the reaction with glycine and washing, fixed material was sonicated in RIPA buffer using a Bioruptor, 4 cycles of 30s on/ 30s off on the high setting.  Sonicated material was supplemented with TritonX-100 to 1%, Sodium DOC to 0.1%, and NaCl to 140mM, and debris was cleared by centrifugation.  Chromatin was aliquoted and stored at -80°C until IP.  For ChIP, 20-30uL of IgG agarose slurry per IP were washed in RIPA buffer and incubated with chromatin overnight.  Bound immunocomplexes were washed 5x with RIPA (140mM NaCl, 10mM Tris pH8, 1mM EDTA pH8, 1% Triton, 0.1% SDS, 0.1% sodium deoxycholate), once with LiCl buffer(250mM LiCl, 10mM Tris pH8, 1mM EDTA pH8, 0.5% NP40, 0.5% sodium deoxycholate), twice with TE, and finally resuspended in TE.  Input and IPs were treated for 30min with RNase at 37°C, then overnight with the addition of proteinase K and SDS (0.5% final), and crosslinks were reversed for 6hrs at 65°C.  IP samples were supplemented with NaCl to 140mM final, and both IP and input samples were extracted with an equal volume of 25:24:1 phenol:chloroform:isoamyl alcohol.  To maximize recovery in IPs, the organic fraction was extracted with TEN140 (TE+140mM NaCl) and pooled with the initial aqueous phase.  All samples were then extracted with an equal volume of 24:1 chloroform:isoamyl alcohol, and precipitated overnight at -80°C with sodium acetate and ethanol, in the presence of glycogen.  The entirety of the precipitated IP-DNA and ~200ng of input DNA were used to create high-throughput sequencing Illumina libraries using the NEBNext ChIP-seq kit (NEB 6240).  Prior to library amplification, size selection was achieved using a 2% agarose gel (Lonza 50111). Libraries for sequencing were prepared using standard Illumina protocols.