title: GSM3135876: B6_3-MII; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted from pools of GV or MII oocytes (n=9-20) collected from a single mother, using the ARCTURUS PicoPure RNA Isolation kit (ThermoFisher #12204-01). Per the protocol oocytes were heat treated in 20 μL PicoPure extraction buffer at 40oC for 30 minutes and then processed immediately or stored at -80oC. During the RNA isolation, the optional DNase treatment was done for 15 minutes at RT (Qiagen #79254). Isolated RNA was eluted into 11 μl of the PicoPure elution buffer and was immediately used or stored at -80oC. Five microliters of the isolated RNA was processed and amplified with SPIA technology using the Ovation RNAseq System V2 (NUGEN #7102-32). Libraries were purified using a Qiagen MinElute Cleanup Kit (Qiagen #28204) and eluted into 22 μl of the provided elution buffer. Purified library concentrations were quantified on a Nanodrop (Thermo Fisher NanoDrop Lite) or Biodrop (uLite) and stored at -80oC. Approximately 1μg of SPIA library sample was diluted into 1X TE for a total volume of 130 μL and was fragmented to an average fragment length of 300 bp on a Covaris shearer. 20 μl of each fragmented SPIA library were then mixed with 14 μl H2O, 4 μl 10X S1 nuclease buffer, and 2 μl S1 nuclease for a 40 μL total reaction volume that was incubated for 30 minutes at RT (Promega # M5761). Starting with an additional bead purification, SPIA libraries were taken through the Ovation Ultralow System V2 1-16 (Nugen #0344-32). Samples were randomized for barcode ligation. Library amplification proceeded for 12 cycles. After the last bead purification, amplified-barcoded libraries were eluted in 30 μL of 1X TE. The first batch of libraries were sequenced on an Illumina HiSeq 2500 system; these libraries were loaded at 65% of standard concentration with PhiX library added up to 10% of the input. The second batch of libraries was sequenced on the HiSeq 4000 system where the input was not decreased and PhiX was loaded only to the standard 1%. 50bp single-end RNAseq
attributes.tag: GEO Accession
attributes.value: GSM3135876