title: GSM7779137: D10 rep 3; Mus musculus; RNA-Seq
library_construction_protocol: RNA was extracted at different time points using the Monarch Total RNA Miniprep Kit (including gDNA removal) and following the manufacturer's instructions. Roughly 150 ng of high quality total RNA (RIN >8) was used for the transcriptomic assays using Illumina's Stranded mRNA Prep ligation according to the manufacturer's instructions. Briefly, oligo(dT) magnetic beads were used to capture and purify poly-adenylated mRNA molecules from total RNA. The purified mRNA was then fragmented and reverse-transcribed to cDNA using random primers. A second strand cDNA synthesis step removed the RNA template while incorporating dUTP in place of dTTP in order to preserve strand specificity. Next, double-stranded cDNA was A-tailed, then ligated to Illumina anchors bearing T-overhangs. PCR-amplification of the library allowed the barcoding of the samples with 10bp dual indexes and the completion of Illumina sequences for cluster generation. Libraries were quantified with Qubit dsDNA HS Assay Kit and their profile was examined using Agilent's HS D1000 ScreenTape Assay. RNA Sequencing and data processing : The quantity and quality of the RNAs were evaluated using Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855) and Agilent RNA 6000 Nano Chips (Agilent Technologies, Cat.# 5067-1511), respectively. Sequencing libraries were prepared following “TruSeq Stranded mRNA Sample Preparation Guide (Part # 15031058 Rev. E)” using the “TruSeq Stranded mRNA Library Prep” kit (Illumina Inc. Cat. # 20020594) and TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (Illumina Inc. Cat. # 20019792). Starting from 700 ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, Cat. # 18064-014) for 10 min at 25C, 15 min at 42C, 15 min at 70C and pause at 4C. cDNA second strand was synthesized with Illumina reagents at 16C for 1 hour. Then, A-tailing and adaptor ligation were performed. Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat. # 5067-4626 ) and quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, Cat. # Q32854). Sequencing was carried out in an Illumina NextSeq2000 using paired-end, dual-index sequencing (Rd1: 59 cycles, i7: 10 cycles, i5: 10 cycles, Rd2: 59 cycles) at a depth of 30 million reads per sample.