title: GSM9213182: 7824 LE; Mus musculus; RNA-Seq
library_name: GSM9213182
library_construction_protocol: Uterine luminal epithelium was isolated by cutting horns into ~4mm segments, opening segments longitudinally to form sheets, shaking sheets in 1mL 1% w/v trypsin in calcium and magnesium free HBSS in 15mL conical tubes for 1.5hr at 4C o a shaking platform, followed by  30 min. shaking at room temperature. 4mL additional HBSS was added to each tube and tubes were shaken vigorously by hand to encourage LE sheets to detach. Contents of each tube was poured onto a 6cm dish. The large chunks were removed with tweezers and placed in new tube for subsequent stromal processing. HBSS was collected from the dish by pipetting and spun at 9,000xG for 5 minutes. Supernatant was removed and remaining pellet was considered isolated LE. To isolate uterine stroma, remaining uterine chunks were incubated in 1mL 0.25% trypsin with 1mg/mL collagenase in HBSS at 37C on a platform shaker at 200rpm for 30 minutes. 3mL additional HBSS was then added to eachy tube, and 5mL serological pipette was used to pipette up and down at least 25 times to release the stromal cells into solution. This was then again moved to a 6cm petri dish, and large chunks (serous and muscle layers) were removed with tweezers and discarded. Remaining solution was passed through a 100um nylon filter (cell strainer), strainer was rinsed with 1mL full serum hESC growth media, and solution was transferred to tubes and spun at 9,000xG for 5 minutes. Pellets were washed once with 500uL full serum hESC growth media to inhibit remaining trypsin, and solutions were spun again at 9,000xG for 5 minutes. Supernatant was removed and remaining pellet was considered isolated uterine stroma. Uterine luminal epithelia and stroma was washed with cold PBS and RNA was isolated using Qiagen RNeasy kit with Dnase treatment. Total RNA integrity was determined using Agilent Bioanalyzer or 4200 Tapestation. Library preparation was performed with 500ng to 1ug of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 12-15 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases.
identifiers.namespace: GEO
identifiers.id: GSM9213182_r1