title: GSM2858142: HA negative RNA-Seq experiment; Mus musculus; RNA-Seq
library_construction_protocol: Nuclear Isolation: Frozen midbrain samples were homogenized in 1 ml ice-cold homogenization buffer [320 mM sucrose, 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH7.6, 0.1 mM EDTA, 0.1% NP40, 0.1 mM PMSF, 1 mM β-mercaptoethanol, 1% BSA, 1:250 RNasin Plus RNase Inhibitor (Clontech)] using a 1 ml Dounce homogenizer (Wheaton); 30 times with pestle A, followed by 30 times with pestle B. After 10 min on ice, the homogenate was filtered with 40 m cell strainer (Fisher) and added 1 ml dilution buffer  [50% OptiPrep density gradient medium (Sigma), 5 mM CaCl2, 3 mM Mg(Ac)2, 10 mM Tris pH 7.6, 0.1 mM PMSF, 1 mM β-mercaptoethanol] and mixed thoroughly with pipette. Loaded 0.5 ml lysate on the top of 0.5 ml 29% iso-osmolar OptiPrep solution (in PBS) in a 1.5 ml centrifuge tube and centrifuged at 6,000 × g for 10 min at 4 °C. After removing the supernatant, the nuclei were resuspended in wash buffer [2.5 mM MgCl2, 1% BSA in PBS, 1:500 RNasin Plus RNase Inhibitor (Clontech)] for immunostaining or FANS.  Nuclear immunolabeling: Isolated nuclei suspended in wash buffer (1% BSA, 2.5mM MgCl2 in PBS) were centrifuged (5 min @1000g) and supernatant was discarded. Nuclear pellet was then resuspended with rabbit anti-HA (1:200, 3724, Cell Signaling, MA) or chicken anti-GFP (1:2000, AbCam) in 200 l wash buffer for 1 hr and centrifuged (5 min @1000g). Supernatant was discarded and pellet was resuspended in 400 l wash buffer for 5 mins and centrifuged again (5 mins @1000g). Nuclear pellet was then resuspended in donkey anti-rabbit 568 (1:500; Invitrogen, Carlsbad, CA) or rhodamine-conjugated donkey anti-chicken secondary (Vector Labs) in 200 l wash buffer for 1h and then centrifuged (5 mins @1000g). Supernatant was discarded and pellet was resuspended in 400 l wash buffer for 5 mins and centrifuged again (5 mins @1000g). Nuclei were resuspended with 1% DAPI in 300 l wash buffer. All steps for nuclear staining were performed at 4°C. RNA-Seq: FANS-sorted nuclei were directly sorted to RLT plus buffer (Qiagen). Total RNA and DNA were prepared from the same samples. Total RNA samples from 400 nuclei were reverse transcribed and amplified using SMARTer Ultra Low Input RNA cDNA preparation kit (Clontech). cDNAs were then fragmented using the Covaris M220 sonicator (Covaris). The fragmented cDNAs were end-repaired, adaptor ligated and amplified using NEBNext Ultra DNA Library Prep Kit for Illumina according to the manufacturer's instruction (New England Biolabs). Single end 100 bp sequencing was performed on a HiSeq2500 sequencer (Illumina).  Low-input DNase-Seq: Low-input DNase-Seq (liDNase-Seq) libraries were prepared as previously described8. Nuclei were collected in 1.5 ml tubes in PBS with BSA during sorting. 500 nuclei were pelleted and resuspended in 36 μl lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 3 mM MgCl2, 0.1% Triton X-100) and incubated on ice for 5 min. DNase I (10 U/μl, Roche) was added to the final concentration of 40 U/ml and incubated at 37 °C for 5 min. The reaction was stopped by adding 80 μl Stop Buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 0.15% SDS, 10 mM EDTA) containing 2 μl Proteinase K (20 mg/ml, Life technologies). Then 20 ng of a circular carrier DNA [a pure plasmid DNA without any mammalian genes was purified with 0.5x Beckman SPRIselect beads (Beckman Coulter) to remove small DNA fragments] was added. The mixture was incubated at 50 °C for 1 hr, then DNA was purified by extraction with phenol-chloroform and precipitated by ethanol in the presence of linear acrylamide (Life technologies) overnight at -20 °C. Precipitated DNA was resuspended in 50 μl TE (2.5 mM Tris, pH 7.6, 0.05 mM EDTA), and the entire volume was used for sequencing library construction.   Sequencing library was prepared using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufactures' instruction with the exception that the adaptor ligation was performed with 0.03 μM adaptor in the ligation reaction for 30 minutes at 20 °C, and that PCR amplification was performed using Kapa Hifi hotstart readymix (Kapa Biosystems) for 8-cycles. The PCR products were purified with x1.3 volume of SPRIselect beads (Beckman Coulter) and then size selected with x0.65 volume followed by x0.7 volume of SPRIselect beads. The samples were amplified again with Kapa Hifi hotstart readymix. The PCR product was purified with x1.3 volume of SPRIselect beads and then size selected with x0.65 volume followed by x0.7 volume of SPRIselect beads. The DNA was eluted in TE. The libraries were sequenced on a Hiseq2500 with single-end 100 bp reads (Illumina).
attributes.tag: GEO Accession
attributes.value: GSM2858142