GSM4818135: CHO_START1; Cricetulus griseus; ncRNA-Seq
[RNA-seq]  RNA was extracted from organs that were homogenized in Trizol LS using an Omni Tissue homogenizer. After incubation at RT for 5 minutes, samples were spun at 21.000g for 3 minutes, supernatant transferred to a new tube and RNA extracted following manufacturerâs instructions. Strand-specific total RNA-seq libraries from ribosomal RNA-depleted RNA were prepared using the TruSeq kit Stranded Total RNA Library kit (Illumina) according to the manufacturer-supplied protocol. [csRNA-seq]  Capped small RNA-sequencing was performed identically as described by (S. H. Duttke et al., 2019). Briefly, total RNA was size selected on 15% acrylamide, 7M UREA and 1x TBE gel (Invitrogen EC6885BOX), eluted and precipitated over night at -80Â°C. Given that the RIN of the tissue RNA was often as low as 2, essential input libraries were generated to facilitate accurate peak calling. csRNA libraries were twice cap selected prior to decapping, adapter ligation and sequencing. Input libraries were decapped prior to adapter ligation and sequencing to represent the whole repertoire of small RNAs with 3â-OH. [sRNA-seq]  Similar to csRNA-seq, without the decapping. [5'GRO-seq and GRO-seq]  Global nuclear run-on sequencing (GRO-seq (Core et al., 2008) and 5âGRO-seq (Core et al., 2008; Lam et al., 2013)) were performed as described below. Nuclei from hamster tissues were isolated as described in (Hetzel et al., 2016). Hamster BMDM nuclei were isolated using hypotonic lysis [10 mM Tris-HCl pH 7.5, 2 mM MgCl2, 3 mM CaCl2; 0.1% IGEPAL] and flash frozen in GRO-freezing buffer [50 mM Tris-HCl pH 7.8, 5 mM MgCl2, 40% Glycerol]. 0,5-1 x 106 BMDM nuclei were run-on with BrUTP-labelled NTPs as described (S. H. C. Duttke et al., 2015) with 3x NRO buffer [15mM Tris-Cl pH 8.0, 7.5 mM MgCl2, 1.5 mM DTT, 450 mM KCl, 0.3 U/Âµl of SUPERase In, 1.5% Sarkosyl, 366 ÂµM ATP, GTP (Roche) and Br-UTP (Sigma Aldrich) and 1.2 ÂµM CTP (Roche, to limit run-on length to ~40 nt)]. Reactions were stopped after five minutes by addition of 750 Âµl Trizol LS reagent (Invitrogen), vortexed for 5 minutes and RNA extracted and precipitated as described by the manufacturer. [GRO-seq]  RNA pellets were fully resuspended in 18 Âµl ddH2O + 0.05% Tween (dH2O+T). Subsequently, 2 Âµl fragmentation mix [100 mM ZnCl2, 10 mM Tris-HCl pH 7.5] was added and the samples incubated at 70Â°C for 15 minutes. RNA fragmentation was stopped by placing samples on ice and addition of 2.5 Âµl 100 mM EDTA p.H. 8.0 and 500 Î¼l cold GRO binding buffer [GRO-BB; 0.25Ãsaline-sodium-phosphate-EDTA buffer (SSPE), 0.05% (vol/vol) Tween, 37.5 mM NaCl, 1 mM EDTA p.H. 8.0]. BrU enrichment was performed using a BrdU Antibody (Sigma B8434-200 Âµl Mouse monoclonal BU-33) coupled to Protein G (Dynal 1004D) beads. Per run on reaction, 3x 20 Âµl of Protein G beads were washed twice in DPBS+0.05% Tween 20 (DPBS+T) and then the antibody coupled in a total volume of 1 ml DPBS+%T under gentle rotation. 1 Âµl of antibody was used per 8 Âµl of beads. After one hour at RT, the beads were collected using a magnet and the supernatant saved for future coupling reactions. Coupled beads were washed twice with GRO-BB prior to use. It is recommended to pre-chill beads to 4áµC prior to the IP as traces of RNase may be observed. RNase activity can be monitored e.g. using IDTâs RNaseAlertÂ®. No crosslinking is required and worked best in our hands followed by DMP >>> Bs3. To each reaction of fragmented NRO-RNA with added GRO-BB, 20 Î¼l equilibrated Î±BrdU beads were added and the samples slowly rotated at 4 Â°C for 80 minutes. Beads were subsequently collected on a magnet and the IP reaction washed twice with 500 Î¼l GRO binding buffer before RNA was eluted by incubation of the beads in 500 Î¼l Trizol LS (Thermo Fisher) under medium agitation for 5 minutes. 120 Î¼l of TET [10 mM Tris-HCl pH 7.5, 1 mM EDTA p.H. 8.0, 0.05 % Tween20] was added to increase the supernatant and RNA extracted as described by the manufacturer. For end-repair and decapping, RNA pellets were dissolved in 8 Âµl TET by vigorous vortexing, heated to 70Â°C for 2 minutes and placed on ice. After a quick spin, 22 Âµl Repair MM [3 Âµl 10x PNK buffer, 15.5 Âµl dH2O+T, 0.5 Âµl SUPERase-In RNase Inhibitor (10 U, Ambion), 2 Âµl PNK (20U, Enzymatics Y904L), 1 Âµl RppH (5U, NEB)] was added, mixed by flicking and incubated at 37Â°C for 1 hour. To phosphorylate the 5âend, 1 Âµl 10 mM ATP was subsequently added and the reactions were incubated for another 45 minutes at 37Â°C (the high ATP concentration is advised to quench RppH activity). Following end repair, 2.5 Âµl 50 mM EDTA was added, reactions mixed and then heated to 70Â°C for 2 minutes before being placed on ice. A second BrdU enrichment was performed as detailed above. For library preparation, RNA pellets were dissolved in 2.75 Âµl TET + 0.25 Âµl Illumina TruSeq 3âAdapter (10 mM), heated to 70Â°C for 2 minutes and placed on ice. 7 Âµl of 3âMM [4.75 Âµl 50% PEG8000, 1 Âµl 10x T4 RNA ligase buffer (NEB), 0.25 Âµl SUPERase-In, 1 Âµl T4 RNA Ligase 2 truncated (200U; NEB)] was added, mixed well by flicking and reactions incubated at 20Â°C for 1 hour. Reactions were diluted by addition of 10 Âµl TET + 2 Âµl 50 mM EDTA, heated to 70Â°C for 2 minutes, placed on ice and a third round of BdUTP enrichment was performed. RNA was transferred as a pellet to PCR strips during the 75% ethanol wash and dried. Samples were dissolved in 4 Âµl TET [10 mM Tris-HCl pH 7.5, 0.1 mM EDTA, 0.05% Tween 20] + 1 Âµl 10 mM reverse transcription (RT) primer. To anneal the RT-primer, the mixture was incubated at 75Â°C for 5 minutes, 37Â°C for 15 minutes and 25Â°C for ~10 minutes.  To ligate the 5â Illumina TruSeq adapter, 10 Âµl 5âMM [1.5 Âµl ddH2O + 0.2% Tween20, 0.25 Âµl denaturated 5âTruSeq adapter (10 mM), 1.5 Âµl 10x T4 RNA ligase buffer, 0.25 Âµl SUPERase-In, 0.2 Âµl 10 mM ATP, 5.8 Âµl 50% PEG8000, 0.5 Âµl T4 RNA ligase 1 (5U; NEB)] was added and reactions were incubated at 25Â°C for 1 hour. Reverse transcription was performed using Protoscript II (NEB) [4 Âµl 5x NEB FirstStrand buffer (NEB; E7421AA), 0.25 Âµl SUPERase-In, 0.75 Âµl Protoscript II (150U; NEB)] at 50Â°C for 1 hour. After addition of 30 Âµl PCR MM [25 Âµl 2x LongAmp Taq 2X Master Mix (NEB), 0.2 Âµl 100 mM forward primer, 2.8 Âµl 5 M Betaine and 2 Âµl 10 mM individual barcoding primer], mixtures were amplified for 13 cycles. PCR reactions were cleaned up using 1.5 volumes of SpeedBeadsâ¢ (GE Healthcare) in 2.5 M NaCl/20%PEG8000 and the libraries size selected on a PAGE/TBE gels to 150â225 bp. Gel slices were shredded by spinning through a 0.5 ml perforated PCR tube placed on top of a 1.5 ml tube. 150 Âµl Gel EB [0.1% LDS, 1M LiCl, 10 mM Tris-HCl pH 7.8] was added and the slurry incubated under agitation overnight. To purify the eluted DNA, 800 Âµl Zymogen ChIP DNA binding buffer (D5205) was added into the 1.5 ml tube containing the shredded gel slice and the Gel EB, mixed by pipetting and the slurry transferred to a ZymoMiniElute column. Samples were first spun at 1000g for 3 minutes, then 10k g for 30 seconds. Flow through was removed, and samples washed with 200 Âµl Zymo WashBuffer (with EtOH). Gel remainders were removed by flicking and columns washed by addition on another 200 Âµl Zymo WashBuffer. Flow through was removed, columns spun dry by centrifugation at 14k g for 1 minute and DNA eluted by addition of 20 Âµl pre-warmed Sequencing TET [10 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 0.05% Tween 20]. Libraries were sequenced on an Illumina NextSeq 500 at using 75 cycles single end. [5' GRO-seq]  RNA pellets were resuspendend in 10 Âµl TET, heated to 70Â°C for 2 minutes and placed on ice. 10 Âµl of dephosporylation MM [2 Âµl 10x CutSmart, 6.75 Âµl dH2O+T, 1 Âµl Calf Intestinal alkaline Pho