title: GSM9330031: D0.5 Esr1f/- Control Luminal Epithelium 3; Mus musculus; RNA-Seq
library_construction_protocol: For each uterus, ¼ of one uterine horn was fixed in 10% formalin and ¼ of the other uterine horn was flash-frozen on dry-ice, designated as “U” (uterus), the remaining ¾ uterine horns connected by the cervix were slit open, immersed in 0.5% dispase (REF#17105-041, gibco, Waltham, MA, USA) in 1xHBSS buffer without calcium or magnesium (REF#1415-052, gibco, Waltham, MA, USA) for 30 min in a 6-cm culture dish, modified from our previous procedure (3-5). To determine the effects of enzymatic digestion process on uterine gene expression, we collected a small piece of digested uterine tissue prior to being processed for LE isolation. The small piece of digested uterine tissue was frozen on dry-ice and designated as “DU” (digested uterus). The LE layer in the remaining uterine tissue was gently scraped. The dislodged LE sheets were collected into a 1.5 ml microcentrifuge tube using a 200 μl pipette tip under a dissection microscope, centrifuged at 2000g for 3 minutes. Upon the removal of the buffer, 1 ml of TRIzol (REF# 15596018, Invitrogen, Carlsbad, CA, USA) was added to the LE pellet, which was gently vortexed to disperse the LE pellet, and kept at -20oC. The U and DU tissues were carefully powdered in liquid N2 using a mortar and pestle, collected into a 1.5 ml microcentrifuge tube, in which 1 ml of TRIzol was added, and upon gentle vortexing to disperse the uterine powder, the tube was kept at -20oC. At the conclusion of tissue collection, the LE, DU, and U samples in TRIzol were processed as one batch per tissue for total RNA isolation. A total amount of 1 µg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations.