title: GSM3325888: cytoplasmic enriched RNA-seq, N2, 23C for four generations; Caenorhabditis elegans; RNA-Seq
library_construction_protocol: Nuclei and cytoplasm factions were obtained using Nuclei PURE Prep kit (Sigma-Aldrich NUC-201) according to manufacture's instruction. All procedure was performed at 4°C. Briefly, worm grinds were re-suspended in 10ml Lysis Solution with 1mM DTT and 0.1% Triton X-100. Proper grinding was confirmed by DAPI staining the lysis and microscopic examination.  The lysis was mixed with 18ml 1.8M Sucrose Cushion, loaded on top of 10ml 1.8M Sucrose Cushion Solution, and ultra-centrifuged at 30,000xg for 45 minutes at 4°C in a SW28 rotor. The clear sucrose cushion layer was collected as cytoplasm fraction and used for RNA extraction immediately.  The pellet of purified nuclei was wash with Nuclei PURE Storage Buffer, and was used as nuclear fraction for RNA extraction immediately.  RNA extraction was performed using the TRIzol Reagent. Ribosomal RNA (rRNA) was removed from the total RNA using RNase H and anti-rRNA oligo mixture. 1.25 µg of anti-rRNA oligos were mixed with 0.5 µg total RNA in 1x Hybridization Buffer (100 mM Tris-HCl pH 7.4, 200mM NaCl) in a final volume of 8 µl.  The sample was denatured at 95°C for 2 minutes, and then cooled at −0.1 °C /sec to 45°C.  Equal volumes of Hybridase Thermostable RNase H (5U/µl,Epicentre) and 10x RNaseH Reaction Buffer (500 mM Tris-HCl pH 7.4, 1 M NaCl, 200 mM MgCl2) were mixed and preheated to 45°C before use. 2 µl of the enzyme mix was added to each reaction mix, which was then incubated at 45°C for 1 hr. To remove DNA oligos, 5 µl TURBO DNase buffer, 3 µl TURBO DNase (Invitrogen), and 32 µl diH2O were added to the reaction mix, followed by 37˚C incubation for 30 minutes.  To purify RNA, 300 µl STOP solution (1 M ammonium acetate, 10 mM EDTA) was added to the reaction mix, followed by phenol/chloroform (1:1) extraction and ethanol precipitation. The resulting RNA (without poly(A) selection) was used to prepare the RNA-seq library using the standard Illumina RNA-seq library construction protocol.