GSM4405114: AF1_96; Mus musculus; RNA-Seq
Individual cells were flow cytometrically purified on a 96 well format into 0.2% Triton X-100 containing RNase inhibitor, dNTPs and oligo-dT primers and stored at -80oC. On thawing, lysates were heated to 72 oC for 3 minutes and subject to reverse transcription, PCR preamplification (26 cycles) and PCR purification.  cDNA library quality was assessed for all samples by qualitative PCR using primers for 18s RNA with an additional check by Bioanalyzer using an Agilent high sensitivity DNA chip on a small subset of libraries. A subset of libraries was quantified using the Qubit dsDNA HS assay kit and an average value used to calculate library dilution to 100-150 pg/ml. cDNA library tagmentation and amplification was performed using the Illumina Nextera XT DNA Library Preparation Kit according to manufacturer's instructions except that all volumes were reduced 4-fold and tagmentation performed at 55oC for 20 minutes. Nextera index and Illumina adapter sequences were incorporated at the amplification stage (N7xx and S5xx). Amplified and indexed libraries were pooled and purified using Agencourt AMPure XP beads at a ratio of 1:0.9 library to beads and washed with 70% ethanol. Two rounds of purification were performed before a final elution in 1.25x total library volume of Nextera Resuspension buffer
GEO Accession
GSM4405114