title: GSM4411349: miRNA_seq_OE_hsa_mir217_C37UA39C_rep1; Homo sapiens; miRNA-Seq
library_construction_protocol: To enrich sRNA <200nt, after RNA extraction by Trizol LS, the total RNA was subjected to gel size selection. For icSHAPE-MaP, The 8 µL purified RNA was ligated with a 3′ linker by incubation with the 3′ ligation mix (6 µL PEG8000, 1 µL 3' linker (10 µM), 1 µL DTT (100 mM), 2 µL 10×ligation buffer, 1 µL T4 RNA ligase KQ (NEB), 1 µL RiboLock) at 25 °C for 2 hrs, followed by inactivation of enzyme at 65°C for 20 min. The 1.2 µL RT primer (10 µM) was added to the 3' ligation mix and annealed to excess 3'linker by 75 °C 5 min, 37 °C 15 min, 25 °C 15 min. The 5' ligation mix ( 3 µL PEG8000, 3 µL 10 mM ATP, 1 µL 10×ligation buffer, 0.5 µL RiboLock, 0.5 µL 5′ linker (Key Resource Table, 20 µM), 1 µL T4 RNA ligase I (NEB)) was added directly and incubated at 25 °C for 2 hrs. The reaction was purified with a RNA concentration kit (Zymo). The 9 µL mutation buffer (50 mM Tris-HCl pH 8.0, 500 µM dNTPs, 75 mM KCl, 10 mM DTT, 6 mM MnCl2, 1 µL RiboLock) was added to the 10 µL purified RNA. The reaction mix was incubated at 42 °C for 2 min. Then 1 µL SuperScript II (Thermo Fisher) was added to the reaction mix. The reverse transcription mix was incubated at 42 °C for 3 hrs. cDNA products were purified with a DNA concentration kit (Zymo).PCR were set up with 20 µL eluted cDNA and PCR reaction mix (0.5 µL P5 primer (Key Resource Table, 20 µM), 0.5 µL P3 index primer (Key Resource Table, 20 µM), 0.4 µL 25×SYBR Green, 20 µL 2×Phusion HF Master Mix (NEB)). PCR was performed in a qPCR machine (Agilent, Mx3000P) to monitor the amplification procedure and was programmed as follows: stage I: 98 °C for 1 min; stage II: 98 °C for 15 s, 65°C for 30 s, 72 °C for 45 s, with limited cycles.PCR products were purified with a DNA concentration kit (Zymo) and a further size selection (150~330 nt) on a 6% Native PAGE gel. For sRNA-seq(40-200nt), 1 µg sRNA was then incubated with the end-repairing mix (2 µL 5×PNK buffer, 2 µL T4 polynucleotide kinase (NEB), 1 µL Fast AP (Thermo Fisher), 1µL RiboLock, 4 µL nuclease-free water) at 37°C for 1 hr. The 3′ ligation mix (6 µL PEG8000, 1 µL 3′ linker (Key Resource Table, preadenylated and blocked at its 3′ end with SpC3, 10 µM), 1 µL DTT (100 mM), 1 µL 10×RNA ligation buffer, 1 µL T4 RNA ligase I (NEB)) was added directly to the end-repairing mix. The ligation condition was 25°C for 3 hrs. The ligation product was size-selected on a 6% Urea-PAGE gel (Thermo Fisher) for ~60-200 nt. Gel purification was performed by crushing the gel and incubating in the RNA elution buffer (20 mM Tris-HCl pH 7.0, 2 mM EDTA pH 8.0, 300 mM NaAc pH 5.2, RiboLock 40 U/mL) with vigorous shaking at 37°C overnight. The eluate was collected by centrifuging through 0.45 um Spin-X columns (Thermo Fisher), followed by ethanol precipitation.   For reverse transcription, 9 µL purified 3′-ligation product was mixed with 1 µL RT primer (Key Resource Table, 10 µM). Samples were heated to 70°C for 5 min in a PCR block, cooled to 25°C by stepping down 1°C every 3 sec and held at 25°C. Afterwards, 8 µL mutation buffer (50 mM Tris-HCl pH 8.0, 500 µM dNTPs, 75 mM KCl, 10 mM DTT, 6 mM MnCl2), 1 µL RiboLock, and 1 µL SuperScript II (Thermo Fisher) were added. The reaction mix was incubated at 25°C for 3 min, and then 42°C for 3 hr. cDNA products were purified by running on 6% Urea-PAGE gel. The following steps, including circularization, PCR amplification and PCR products purification, were done as previously described. For miRNA-Seq (~20 nt), library construction, Illumina sequencing and data analysis were conducted as previously described (Gu et al., 2012), except for the following modification. Reverse transcription (RT) was set up with RNAs ligated with adaptors, 1 µL RT primer, 1×SSIV buffer, 500 µM dNTP, 5 mM DTT, 1 U/µL RiboLock, and 1 µL SuperScript IV (Thermo Fisher) in a total volume of 20 µL. The reaction was programmed as follows: 25°C for 3 min, 42°C for 10 min, 50°C for 30 min, and 80°C for 10 min. RT products were then ethanol precipitated and dissolved in 10 µL H2O. To set up PCR reactions, 5 µL of RT products were mixed with 1×Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB), 200 µM Solexa Forward and Reverse primers in a total volume of 50 µL. PCR was programmed as follows: stage I: 98°C for 30 s; stage II: 98°C for 10 s, 52°C for 30 s, 72°C for 15 s, repeating for total 22 cycles; Stage III: 72°C for 5 min. Multiplex sequencing was performed on an Illumina NextSeq 500 machine or on a MiSeq machine (1X36-bp) at the Stanford Functional Genomics Facility.
attributes.tag: GEO Accession
attributes.value: GSM4411349