title: GSM7830690: Rln_2hr_Replicate1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from myometrial tissue using a RNeasy Plus Mini Kit (Qiagen), according to manufacturer's instructions.  Myometrial tissue was homogenized in RLT buffer with a cooled blade homogenizer (Power Gen25; Fisher). The samples were centrifuged to remove debris and passed through a gDNA eliminator column, followed by RNA binding column. After multiple washes, total RNA was eluted with RNase-free water.  The eluted RNA was assayed for quality by electrophoresis (Agilent Technologies, Santa Clara, CA, USA).  Only samples with RIN values greater than 8 were included for RNA-sequencing. RNA-seq libraries from mouse myometrial tissue were prepared as follows using a protocol described previously (Zhong et al., 2011).  Briefly, biological replicates were generated by pooling 1.5 mg of total RNA from five animals for a total of 7.5 mg of total RNA per replicate.  A total of ten animals were used for each treatment time point.  Polyadenylated RNA was isolated from total RNA using Oligo(dT)25 Dynabeads (Invitrogen) and reverse transcribed using SuperScript III Reverse Transcriptase (Invitrogen).  Second strand synthesis was performed using DNA polymerase I.  Complementary DNA ends were repaired, tailed with dATP, and ligated with barcode adapters for Illumina sequencing platform.  Ligated libraries were selected for average size 270 bp, PCR amplified for 9 cycles, and sequenced on an Illumina NextSeq 500 in a 75 bp single-end format in the McDermott Center Next Generation Sequencing Core at UT Southwestern Medical Center to a total read count of ~40 M per condition.