title: GSM1627067: nuclearRNAseq_exercise1; Rattus norvegicus; RNA-Seq
library_construction_protocol: End repair or second strand synthesis reactions were purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 15 µL dA-tailing mix (NEBNext dA-Tailing Module; New England Biolabs). This mix was incubated at 37 °C for 30 min, purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 14 µL of pre-annealed barcoded adapters at a 20× molar amount relative to the estimated amount of dA-tailed DNA. Next, 4 µL 5× Ligation Buffer and 2 µL Quick Ligase (NEBnext Quick Ligation Module; New England Biolabs) were added, and the reaction was incubated for 15 min at 37 °C.  These reactions were again purified using Agencourt AMPure XP (Beckman Coulter) and eluted in 50 µL water for ChIP sequencing, or 49 µL TE buffer and 1 µL USER enzyme mix (New England Biolabs) for nuclear RNA sequencing. The latter mix was incubated at 37 °C for 30 min and at 95 °C for 5 min. To amplify these libraries, 50 µL Phusion High-Fidelity PCR Master Mix with HF Buffer (Thermo Scientific, Loughborough, UK) was added to each sample, together with primer 1 and 2 oligonucleotides to a final concentration of 500 nM. Each reactions was distributed between 4 PCR tubes and PCR was performed in a thermal cycler using the following program: initial denaturation at 98°C for 30 seconds, amplification over 18 cycles of 98 °C, 65 °C and 72 °C for respectively 10, 30 and 30 seconds, and a final extention at 72 °C for 7 min. PCR reactions were purified using the QIAquick PCR purification kit, eluted in 25 µL, mixed with 10 µL loading buffer (200 mg/mL Ficoll 400, 15 mg/mL Orange G and 2× Tris/Borate/EDTA buffer) and loaded on a 1.7% TBE agarose gel, prestained with SYBR safe DNA gel stain (Life Technologies). DNA was separated by electrophoresis at 4V/cm for 2 hours at 4 °C, and 200 to 400 bp fragments were excised and purified using the QIAquick Gel Extraction Kit (QIAGEN) as per the manufacturer’s instructions. DNA fragment size was verified on a 2100 Bioanalyzer Instrument using a High Sensitivity DNA Analysis Kit (both from Agilent Technologies Inc., Santa Clara, USA), and DNA concentration was verified using the Quant-iT PicoGreen dsDNA Assay Kit (Life Technologies). nuclei sorting protocol: To minimize handling times and to control potential inter-experimental artefacts, nuclei were isolated and sorted from 2 left ventricular wall samples per experiment, one hypertrophic sample and one corresponding control sample. Methods for nuclear isolation were based on a previously described method.To minimize handling times and to control potential inter-experimental artefacts, nuclei were isolated and sorted from 2 left ventricular wall samples per experiment, one hypertrophic sample and one corresponding control sample. Methods for nuclear isolation were based on a previously described method. All buffers were precooled, supplemented with dithiothreitol (1 µM), actinomycin D (1mg/L), cOmplete protease inhibitor (PI) cocktail (Roche; 0.5 µL/mL) and SUPERase.In (Ambion; 200 U/mL), and buffered with 10 mM Tris-HCl to pH 8.0. Frozen hearts where thawed in 10 mL lysis buffer (0.32 M sucrose, 5 mM CaCl2, 5 mM EDTA, 0.5 mM EGTA, 3 mM Mg(CH3COO)2) and homogenized using an IKA ultra-Turrax (20,000 RPM, 15 sec). After addition of 30 mL lysis buffer, the homogenate was dounced 10× on ice, filtered consecutively over mesh, a 100 µm and a 70 µm cell strainer and spun for 10 minutes (700×G; 4°C). The resulting pellet was dissolved in 10 mL centrifuge buffer (2.1 M sucrose, 3 mM Mg(CH3COO)2, and 5 mM EDTA), and superimposed upon 5 mL centrifuge buffer in an autoclaved Thinwall Ultra-Clear Tube (Beckman Coulter, High Wycombe, UK). Tubes were loaded in a precooled rotor (SW32.1Ti) and spun for 60 minutes (26,000×G; 4°C) in an OPTIMA MAX Ultracentrifuge (both from Beckman Coulter). The resulting pellet was dissolved in 1.5 mL sorting buffer (0.43 M sucrose, 70 mM KCl, 2 mM MgCl2 and 5 mM EDTA) and labelled for 1 hour on ice using 3 µL anti-PCM1 antibody (HPA023374) that was pre-incubated for 15 minutes with 2 µL Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody (Life Technologies). Nuclei were sorted on an Influx streaming air high-speed cell sorter (BD Biosciences, Oxford, UK), into Protein LoBind tubes (Eppendorf, Stevenage, UK) on a cooled tube holder. For comparison of unsorted and sorted nuclei, the input samples corresponded to a 350 µL aliquot of the original homogenized sample, and the nuclear samples corresponded to 400,000 nuclei not gated for PCM1 staining.  All buffers were precooled, supplemented with dithiothreitol (1 µM), actinomycin D (1mg/L), cOmplete protease inhibitor (PI) cocktail (Roche; 0.5 µL/mL) and SUPERaseIn (Ambion; 200 U/mL), and buffered with 10 mM Tris-HCl to pH 8.0. Frozen hearts where thawed in 10 mL lysis buffer (0.32 M sucrose, 5 mM CaCl2, 5 mM EDTA, 0.5 mM EGTA, 3 mM Mg(CH3COO)2) and homogenized using an IKA ultra-Turrax (20,000 RPM, 15 sec). After addition of 30 mL lysis buffer, the homogenate was dounced 10× on ice, filtered consecutively over mesh, a 100 µm and a 70 µm cell strainer and spun for 10 minutes (700×G; 4°C). The resulting pellet was dissolved in 10 mL centrifuge buffer (2.1 M sucrose, 3 mM Mg(CH3COO)2, and 5 mM EDTA), and superimposed upon 5 mL centrifuge buffer in an autoclaved Thinwall Ultra-Clear Tube (Beckman Coulter, High Wycombe, UK). Tubes were loaded in a precooled rotor (SW32.1Ti) and spun for 60 minutes (26,000×G; 4°C) in an OPTIMA MAX Ultracentrifuge (both from Beckman Coulter). The resulting pellet was dissolved in 1.5 mL sorting buffer (0.43 M sucrose, 70 mM KCl, 2 mM MgCl2 and 5 mM EDTA) and labelled for 1 hour on ice using 3 µL anti-PCM1 antibody (HPA023374) that was pre-incubated for 15 minutes with 2 µL Alexa Fluor 488 Goat Anti-Rabbit IgG (H+L) Antibody (Life Technologies). Nuclei were sorted on an Influx streaming air high-speed cell sorter (BD Biosciences, Oxford, UK) using a gating strategy as described.15 Post-sorting flow cytometric analyses were done using FlowJo (v9.6.3) and Bioconductor’s flowCore package.52 Nuclei were sorted into Protein LoBind tubes (Eppendorf, Stevenage, UK) on a cooled tube holder. For comparison of unsorted and sorted nuclei, the input samples corresponded to a 350 µL aliquot of the original homogenized sample, and the nuclear samples corresponded to 400,000 nuclei not gated for PCM1 staining. DNA libraries were sequenced on an Illumina HiSeq 1000 (Illumina, Essex, UK) using either the default RTA software, or a custom program that consisted of annealing of Illumina Read 1 sequencing primer, 4 dark cycles, 46 light cycles, denaturation, annealing of Illumina Read 1 sequencing primer and 5 light cycles to read the indexing barcode. The latter modification was introduced because the low complexity of the first bases from the indexing barcode yielded incorrect cluster definitions and consequently lowered the number of high quality reads.
attributes.tag: GEO Accession
attributes.value: GSM1627067