Sick Of Every Vasopressin Receptor Scoops? We're There For You Personally
Human fibroblasts, and siRNA-transfected HEK293 cells were harvested 48?hr post-transfection, and total RNA isolated using TRIzol reagent (Invitrogen). OligodT magnetic dynabeads were then used to isolate the mRNA fraction and libraries for sequencing were then prepared using the Illumina TruSeq mRNA Prep Kit. For small RNA sequencing, total RNA from human fibroblasts was purified with the MirVana kit (Ambion) in order to isolate the?Vasopressin Receptor was then ethanol precipitated to recover RNA and these samples were then used as the input for the Illumina small RNA Prep Kit. All next generation sequencing was performed on the Illumina GA2 platform. All experiments were performed in quadruplicates. RNA was isolated from human fibroblasts using TRIzol and cDNA synthesis performed using the SuperscriptIII reverse transcriptase kit (Invitrogen). Unless otherwise stated, qPCR was then performed using TaqMan assay sets purchased from Applied Biosystems as per manufacturers recommendations. TaqMan probe sets used were CACNG7: Hs00259061_m1, CACNG8: Hs01100182_m1, NSUN2: Hs00214829_m1, and GAPDH (4326317E) as an internal control. Total RNA from NSUN2+/? and NSUN2?/? dermal fibroblasts was isolated using TRIzol (Invitrogen). RNAs of?selleck compound gels (Invitrogen) and gel slices containing10 to 40 base pairs were excised and incubated with RNA free water for 2?hr at 4��C to elute RNA. RNA was then purified using spin-X centrifuge tube filters (0.22?��M) (Costar) and ethanol precipitated over night at ?20��C. Small RNA Reverse transcription and qPCR was performed as previously described ( Shi and Chiang, 2005). Briefly, polyA tails were added to small RNAs using poly(A) polymerase (Ambion). The poly(T) primer 5��-GCG AGC ACA GAA TTA ATA CGA CTC ACT ATA GG(T)12VN-3�� was then used for cDNA synthesis. QuantifastSYBR? green master mix (QIAGEN) was used for QPCR reactions. The forward primers were: miR-16 (5��-TAG CAG CAC GTA AAT ATT GGC G-��3), miR-150 (5�� TCT CCC AAC CCT TGT ACC AGT G 3��), miR-92b (5�� AGG GAC GGG ACG CGG TGC AGT G 3��), svRNA1 (5��-TGT CTG GGT TGT TCG AGA CCC GCG GGC-3��), Protein Tyrosine Kinase inhibitor svRNA4 (5��-CGA GAC CCG CGG GCG CTC TCC AGT CCT TTT-��3). The reverse primer for all reactions was: 5��-GCG AGC ACA GAA TTA ATA CGA C-3��. Nsun2??/? dermal fibroblasts were lysed in a buffer consisting of 1% NP-40, 25?mM Tris-HCl at pH 7.4, 30?mM NaCl and 10?mM MgCl2 for 30?min on ice and lysates were then pooled and divided equally between samples. 100 pmol of synthetic VTRNA1.1 RNA that either was unmodified or contained the m5C modification at position 69 (Thermo Scientific) was then added to samples, whereas no oligo was added to control samples.
