title: GSM3011768: C8: LN3 CD69+ RNA-seq; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-seq: 250 cells per subset were sorted using a FACSAriaII (BD Biosciences) directly into lysis buffer provided with the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) and snap frozen. RNA-seq:  RNA-seq libraries were prepared using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara) according to manufacturer's protocols. Briefly, 3' SMART-Seq CDS (oligo-dt) Primers were hybridized to the poly(A) tail of all the mRNA and reverse transcribed with the SMARTScribe RT enzyme. cDNA was pre-amplified using SeqAmp DNA polymerase and frozen. Amplified material was purified using Agencourt AMPure XP beads (Beckman) and cDNA quantity was assessed on a Qubit 3.0 (Thermo) and fragment size was evaluated on a 2100 BioAnalyzer (Agilent). The PCR products were next indexed using the Nextera XT DNA Library Prep Kit (Illumina) according to manufacturer's protocol. Briefly, products were tagmented using the Amplicon tagment mix, containing Tn5 transposase, and indexed using Nextera index 1 (i7) and index 2 (i5) primers. The libraries were again cleaned-up with Agencourt AMPure XP beads, pooled, quantified and sequenced across 75 base pairs (bp) using a paired-end strategy with a 150-cycle high output flow cell on a NextSeq 550 (Illumina).
identifiers.namespace: GEO
identifiers.id: GSM3011768