GSM7747167: Follicle small, Patient 1, 13; Homo sapiens; miRNA-Seq
Cryopreserved cortex tissue tubes were thawed by placing tubes into 37°C water bath for ~1 min. Tissue pieces were transferred into thawing media solutions with 10mg/ml HSA (Vitrolife) and decreasing sucrose concentrations (media I, II and III). All incubation steps were 10min. Thawed cortex tissue pieces were mechanically and enzymatically dissociated using McIIwain Tissue Chopper and digestion media containing McCoy's (Thermo Fisher Scientific) medium with 1mg/mL HSA (Vitrolife), 1x GlutaMAX (Thermo Fisher Scientific), 1x Insulin-Transferrin solution (Thermo Fisher Scientific), 40μg/mL Liberase (Sigma-Aldrich), 0.4mg/mL Collagenase IV (Thermo Fisher Scientific), and 0.2mg/ml DNase I (BioRad). The chopped pieces were then incubated in a digestion medium containing Neutral Red (50mg/mL, Sigma-Aldrich) in +37°C shaking for 30–50min and the digestion was stopped using termination medium containing McCoy's with 10% HSA (Thermo Fisher Scientific and Vitrolife). The proportion of viable (red) follicles of all follicles was evaluated under a stereomicroscope (Leica S9D) and only viable follicles were picked. Individual follicles were photographed under the microscope before they were lysed in 4ul of lysis buffer containing 0.2% Tween 20 (Merck), 2U/ul RNase inhibitor (Invitrogen) in RNase-free water (Invitrogen) and stored at -80°C until library preparation. Starting amount of small RNA in library preparation was 2µl of follicle cells lysate. Small RNA libraries were prepared with QIAseq miRNA Library Kit (QIAGEN) according to the manufacturer's protocol. Final libraries were separated and excised from 5% TBE gels (Bio-Rad) after staining with 1X SYBR Gold stain (Thermo Fisher Scientific). Gel pieces containing the small RNA libraries were crushed with pellet pestles (Fisher Scientific). 300µl RNase free water (Thermo Fisher Scientific) was added to the gel debris and rotated for 2h at room temperature (RT) to elute miRNA libraries. Eluate and gel debris were transferred to the Spin X centrifuge tube filter (Merck) and centrifuged 2min at 16 000g. Thereafter 2µl glycogen (Thermo Scientific), 30µl 3M NaOAc (Thermo Fisher Scientific), 1µl 0.1x Pellet Paint (Merck) and 975µl of cold 100% ethanol (Naxo) were added to the eluate, and centrifuged for 20min at 20 000g at 4°C. Pellet was washed with 500µl of 70% ethanol and centrifuged for 2min at 20 000g. The final libraries were resuspended in 7µl of resuspension buffer (PerkinElmer). The size of libraries was estimated with Agilent DNA High Sensitivity chips on the Agilent 2100 Bioanalyzer system (Agilent Technologies). Library concentrations were measured using Qubit High Sensitivity Assay kit (Thermo Fisher Scientific) before pooling in equimolar amounts. Single-end sequencing of 75bp with single index (6bp) length was performed on NextSeq 500 platform with NextSeq HIGH75 kit or NextSeq MID150 kit (Illumina).