GSM1440304: mRNA-seq Basal Replicate 1; Homo sapiens; RNA-Seq
Total RNAs were extracted with acid-phenol:chloroform, pH 4.5 with isoamyl alcohol, 25:24:1. Replicate, strand-specific total mRNA-seq libraries were prepared in parallel using the two-linker ligation protocol as described in Levin et al. Nat Methods 2010 (PMID: 20711195). RNA pellets were air-dried briefly, resuspended in 10 µL of PNK mix (70mM Tris pH 7.6, 10mM MgCl2, 5mM DTT, 1mM ATP, 1U/µL T4 polynucleotide kinase, 1U/µL SUPERase·In), and incubated at 37°C for 30 min. The reaction was combined with 90µL H2O and 100µL Phenol:Chloroform:Isoamyl Alcohol 25:24:1, mixed well, and spun for 5 min at 20,000Xg. The liquid phase was mixed with 12.5µL of 3M NaOAc, 250µL of 100% ethanol, 1µL of 15mg/mL glycoblue and precipitated for 2 hr at -80°C. RNAs were collected by centrifugation for 20 min at 20,000Xg at room temperature, followed by 2 washes with cold 75% ethanol. RNA pellets were resuspended in 10µL of ligation mix (50mM Tris pH 7.5, 10mM MgCl2, 10mM DTT, 1mM ATP, 0.1mg/mL BSA, 2µM 5’RNA linker, 1U/µL T4 RNA ligase, 1U/µL SUPERase•In, 10% DMSO) and incubated at 15°C for 2 hr. Ligation reaction was terminated by adding 10µL of 2X formamide gel loading buffer, heated for 2 min at 70°C, and then quickly chilled on ice. Samples were loaded onto a 6% TBE UREA gel and run at 150 V for 45 min. After staining with 1X Sybr Gold Stain, a gel piece corresponding to 70-90nt RNA (80-100nt ssDNA) was excised, crushed, and soaked in 400µL of 0.3M NaOAc overnight at room temperature. After removing gel pieces, the solution was combined with 1mL of 100% EtOH and 1µL of 15mg/mL glycoblue and precipitated for 2 hr at -80°C. RNAs were collected by centrifugation for 20 min at 20,000Xg at room temperature, followed by two washes with cold 75% ethanol. After brief drying, RNAs were resuspended in 15µL of H2O. 10μL of ligated RNA was combined with 2μL of 5µM RT primer, heated at 65°C for 5 min, and then quickly chilled on ice, followed by the addition of 1µL 10mM dNTP, 1μL of 0.1 M DTT, 4μL of 5X First strand buffer, 1μL of SUPERase•In (20 U/µL) and 1 µL of SuperScript III Reverse transcriptase (200 U/µL). RT reaction was kept at 50°C for 45 min, 55°C for 15 min and 90°C for 5 min. A test PCR was performed with 2.5 µL of RT product in 50 µL PCR mix: 1X AccuPrime PCR buffer I, 0.5 µM P5 long primer, 0.5 µM P7 primer, 0.2 µL AccuPrime Taq High Fidelity (5 U/µL). PCR was carried out with an initial 3 min denaturation at 98°C, followed by 14-22 cycles of 80 sec denaturation at 98°C, 90 sec annealing and extension at 65°C, and termination with a final 5 min extension at 65°C. 15 µL PCR product was collected after 14, 18, and 22 cycles and analyzed on a 10% TBE gel at 150 V for 1 hr to determine the optimal amplification cycles (the lowest cycle number required to generate 96-116 bp amplicons detected by Sybr Gold staining). A 50 µL PCR reaction was carried out with the determined cycle number. Amplicons were purified using DNA clean and concentrator-5, run on 10% TBE gels at 150 V for 1 hr and stained with Sybr Gold. A gel piece corresponding to 96-116 bp DNA was excised, crushed, and soaked overnight in 400 µL 0.3 M NaOAc at room temperature. After removing gel pieces, the solution was combined with 1 mL of 100% EtOH and 1 µL of 15 mg/mL glycoblue and precipitated for 2 hr at -80°C. DNAs were collected by centrifugation for 20 min at 20,000Xg at room temperature, followed by two washes with cold 75% ethanol. After brief drying, amplicons were resuspended in 20 μl of H2O. Purified amplicons (5 μl) were used to seed a second round of PCR in 50 μl: 1× Accu- Prime PCR buffer I, 0.5 μM Illumina Primer A, 0.5 μM Illumina Primer B, 0.2 μl AccuPirme Taq High Fidelity for 6 to 12 cycles. Second PCR amplicons were purified with DNA clean and concentrator-5.
GEO Accession
GSM1440304