title: GSM8487379: hTERT-HM_S118Emutantcells -E2_Replicate1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from myometrial tissue or cultured hTERT-HM-ER⍺ cells using an 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).  hTERT-HM-ER⍺ cells were collected and lysed in RLT buffer.  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 in subsequent analyses. RNA-seq libraries from myometrial tissue were prepared as follows. 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. RNA-seq experiment was performed in two biological replicates of wild-type and S118E mutant hTERT-HM cells treated with Veh, E2, Rln and E+R at 1h. 0h treatment is taken as the control samples.