GSM7747123: Follicle, half, Patient 2, 11; Homo sapiens; RNA-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 RNA in library preparation was 2µl of follicle cells lysate. Before removing 2µl of cell lysate from tube follicles were lysed at 72°C for three minutes. Full-length RNA libraries were prepared according to the Smart-seq2 protocol  with small modifications as follows.  To the cell lysate placed on a 96-well plate (Eppendorf) 2µl lysis buffer together with 1µl 10mM dNTP mix and 1µl freshly diluted 10 µM Oligo-dT30VN (Metabion) were added. In reverse transcription reaction, RNase-free (Invitrogen) water volume was 0.59 µl to reach reaction volume of 6 µl which then was added to the 4µl cell lysate to obtain 10µl reaction volume. cDNA preamplification included 5.5µl 5x Phusion GC buffer and 0.55µl Phusion enzyme (2U/µl) (Thermo Fisher Scientific), 0.825µl DMSO (AppliChem), 0.55µl 10 mM dNTP mix and 0.275µl freshly diluted 10 µM ISPCR (Metabion) primer in 8.8µl RNase-free water (Invitrogen). PCR products were purified using AMPure XP beads (Beckman Coulter) in 1:0.9 ratio. DNA concentrations and DNA profile were measured Qubit HS DNA assay (Invitrogen) and Agilent HS DNA assay (Agilent Technologies), respectively. Tagmentation reaction was carried out by using Illumina Nextera XT DNA preparation kit (Illumina). Tagmentation reaction included 2µl ATM and 4µl TD buffer. To the tagmentation reaction 1ng of cDNA was added with final volume of 10µl.  Amplification of adapter-ligated fragments was carried out using 6µl Nextera PCR Master Mix, 2µl i5 primer, and 2µl i7 primer (both from TAG Copenhagen) using 12 cycles to amplify the DNA. Amplification reaction was purified using AMPure XP beads in 1:0.9 ratio. Final DNA library was quality controlled using Qubit HS DNA assay (Invitrogen) and Agilent HS DNA kit (Agilent Technologies). Libraries were sequenced in equimolar amounts using Single-end 76 bp with dual index (8 bp) sequencing on NextSeq 500 platform with NextSeq HIGH75 kit (Illumina).