GSM2665691: Ctrl_rep1; Homo sapiens; RNA-Seq
Tissue samples from fifteen women with pelvic endometriosis were used in the study. Four patients were recruited for Ion Torrent AmpliSeq RNA-sequencing analysis. Independently, an additional eleven patients were involved in the study to perform real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) analysis for the validation of sequencing data. Clinical characteristics of the patients are shown in Supplemental Table 1. All women were Caucasian. The patients ranged in age from 24 to 39 years (average age 31.5±1.1 years). Seven and six patients were seen with primary and secondary infertility, respectively. Two patients did not have reproductive history. The same patient provided both eutopic and ectopic endometrium samples. Endometriotic lesions from the pelvic region were removed at laparoscopy. Biopsy tissues of eutopic endometrium from the patients were collected by hysteroscopy. Laparoscopy and hysteroscopy procedures were performed during the same surgical intervention in the mid secretory phase of the menstrual cycle. No patients were taking medications and receiving hormone therapy at the time of the surgery or in the previous six months. After surgical resection, the samples were immediately placed in an RNAlater (Ambion, UK), incubated at 4˚C a day and then stored at -70˚C until used. Among the fifteen patients, two women had stage I, nine – stage II, two – stage III, and two - stage IV endometriosis. The severity of endometriosis was classified into stage according to the revised American Society for Reproductive Medicine classiﬁcation of endometriosis.   Total RNA was extracted from tissues with the PureLink RNA Mini Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer’s protocol and quantiﬁed by Qubit 2.0 Fluorimeter with Qubit® RNA HS Assay Kit (Invitrogen, Carlsbad, CA, USA). Integrity of the RNA was assessed on 2200 TapeStation Instrument with High Sensitivity RNA ScreenTape and High Sensitivity RNA ScreenTape Reagents (all purchased from Agilent Technologies, Santa Clara, CA, USA). Purified RNA samples were stored at ‑70˚C until used. The sequencing libraries were prepared with the Ion AmpliSeq RNA Library Kit (Thermo Fisher Scientiic, Inc.) using a custom Ion Torrent AmpliSeq Panel according the manufacturer's protocol. The construction of the library involved the following steps: reverse transcription reaction, multiplex polymerase chain reaction (PCR), digestion primers, ligation of the adapters and the libraries amplification. Briefly, for each sample, 10 ng of total RNA was used as the starting template to synthesize single strand cDNA. For this RNA samples were mixed with 5X VILO RT Reaction Mix and 10X SuperScript III Enzyme Mix (all taken from Ion AmpliSeq™ RNA RT Module), incubated at 42˚C for 30 min and 85˚C for 5 min, and then cooled on ice. The cDNA was amplified using custom primers, described as above, and Ion AmpliSeq™ HiFi master mix (Ion AmpliSeq™ Library Kit 2.0). Then multiplex PCR was performed under the following cycling conditions: denaturation at 99 °C for 2 min; 16 cycles of amplification for 15 sec at 99 °C and for 4 min at 60 °C; then 4˚C for hold. Following target amplification the samples were treated with 2 μL FuPa reagent (Ion AmpliSeq™ Library Kit 2.0) to partially digest primer sequences and phosphorylate the amplicons at 50°C for 10 min, followed by 55°C for 10 min, then 60°C for 20 min. The amplicons were then ligated to adapters (Ion AmpliSeq™ Library Kit 2.0) with the diluted barcodes of the Ion Xpress™ Barcode Adapters kit (Thermo Fisher Scientific, Inc.) for 30 min at 22°C then 72°C for 20 min. To remove high molecular-weight DNA and primers, the amplicons with adapters were purified using Ion AmpliSeq™ RNA Dynabeads® Cleanup Module. The subsequent PCR were performed using purified amplicons with Library Amplification Primer Mix and Platinum PCR SuperMix High Fidelity (Ion AmpliSeq™ Library Kit 2.0). Cycling conditions were as follows: 98˚C for 2 min, 5 cycles of 98˚C for 15 sec and 60˚C for 1 min, then 10˚C for hold. Amplified libraries were purified using Ion AmpliSeq™ RNA Dynabeads® Cleanup Module. For assessment of the yield, size distribution and molar concentration of libraries after cleanup, the samples were run on 2200 TapeStation Instrument with High Sensitivity D1K ScreenTape and High Sensitivity D1K Reagents (all purchased from Agilent Technologies, Santa Clara, CA, USA). All libraries were diluted to 100 pM according to the manufacturer's protocol (Ion AmpliSeq™ RNA Library Kit, Thermo Fisher Scientific, Inc.).
GEO
GSM2665691
GEO Accession
GSM2665691