title: GSM4041796: ILC_ST2_neg_CD127_pos_Rep2; Mus musculus; RNA-Seq
library_construction_protocol: ILC RNA was extracted using RNA Easy Plus Micro kit (Qiagen) according to the manufacturer's protocol.  Prior to RNA seq library preparation, Samples were quantified as described previously (Rosales et al, 2018) and quality of RNA assessed by Fragment Analyzer (Advance Analytical). All samples had a RNA integrity number (RIN)  > 7.5. Purified total RNA (≈5 ng) was amplified following the Smart-seq2 protocol (Picelli et al, 2013, Rosales et al, 2018). Briefly, mRNA was captured using poly-dT oligos and directly reverse-transcribed into full-length cDNA using the described template-switching oligo. cDNA was amplified by PCR for 15 cycles and purified using AMPure XP magnetic bead (0.9:1 (vol:vol) ratio, Beckman Coulter).  From this step, for each sample, 1 ng of cDNA was used to prepare a standard NextEra XT sequencing library (NextEra XT DNA library prep kit and index kits; Illumina).  Barcoded Illumina sequencing libraries (NextEra; Illumina) were generated utilizing an automated platform (Biomek FXP, Beckman Coulter).  Both whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability.  Quality control steps were included to determine total RNA quality and quantity, the optimal number of PCR preamplification cycles, and fragment library size.  None of the samples failed quality controls. libraries were pooled at equimolar concentration, loaded and sequenced on the Illumina Sequencing platform (HiSeq2500, or NovaSeq6000).  Libraries were sequenced to obtain more than 10 million 50-bp single-end reads (HiSeq Rapid Run Cluster and SBS Kit V2; Illumina) mapping uniquely to mRNA reference.
attributes.tag: GEO Accession
attributes.value: GSM4041796