title: GSM2199331: TSPAN8-low subpopulation of P6 ID4-EGFP+ - sample 3; Mus musculus; RNA-Seq
library_construction_protocol: Sorted P6 ID4-EGFP+ spermatogonia and subpopulations defined by antibody labeling for TSPAN8, EPHA2, or PVR were used for RNA-seq analysis of transcriptomes. For this purpose, sorted cells were pelleted, counted and subjected to direct cDNA synthesis using SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech Laboratories, Mountain View, CA) per manufacturer recommendations and 9 cycles of amplification. Amplified cDNAs from each sample were purified using Agencourt AMPure XP Pure magnetic beads (Beckman Coulter Life Sciences, Indianapolis, IN) and pre-qualified for library construction using a 2100 Bioanalyzer and High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA). Using 250pg input cDNA, we performed Nextera XT dual-index library preparation with modifications from manufacturer recommendations. Specifically, tagmentation was performed with 2.5ul Tagment DNA buffer, 1.25ul Amplification Tagment Mix, and 1.25ul cDNA for exactly 10 minutes at 55°C, ramp to 10°C, and immediate addition of 1.25ul NT buffer.  Additionally, PCR amplification with index primers (separate index per sample) was performed with the entire 6.25ul of Tagmentation reaction mix plus 3.75ul Nextera PCR Mix using the recommended cycling conditions except that the extension time was 60 seconds. Libraries were quantified with Qubit fluorometry (ThermoFisher Scientific) and qualified for fragment size and distribution by 2100 Bioanalyzer with High-sensitivity DNA reagents (522 ± 6 bp). Libraries were pooled at equal concentrations and subjected to four lanes of rapid mode Illumina HiSeq2500 sequencing (paired-end 100bp) at the University of Texas Southwestern Medical Center Genomics and Microarray Core.
attributes.tag: GEO Accession
attributes.value: GSM2199331