alias: GSM3305230
title: GSM3305230: CelSeq2_Mixture 383 samples; Homo sapiens; RNA-Seq
library_construction_protocol: For the three cell lines, cells were dissociated into single cell suspensions in FACS buffer and sorted for 9-cell-mixture and single cell experiment (see below for sorting strategy).   The remaining cells were centrifuged and frozen at -80C for later RNA extraction. The RNA was then extracted using a Qiagen RNA miniprep kit. The amount of RNA was quantified using both Invitrogen Qubit fluorometric quantitation and an Agilent 4200 bioanalyzer to get an accurate estimation. The extracted RNA was then diluted to 60 ng\ul and then mixed in different proportions, according to the study design. The different mixtures were further diluted to create an RNA series that ranged from 3.75pg to 30pg that was dispensed into CEL-seq2 and SORT-seq primer plates using a Nanodrop II dispenser. Prepared RNA mixture plates were sealed and immediately frozen upside down at -80C. For CEL-seq2, Single cells were flow sorted into chilled 384-well PCR plates containing 1.2ul of primer/ERCC mix using a BD FACSAria III flow cytometer. Sorted plates were sealed and immediately frozen upside down at -80C. Then these plates, together with the mRNA mixture plates, were taken from -80C and processed using an adapted Cel-Seq2 protocol with the following variations. The second strand synthesis was performed using NEBNext Second Strand Synthesis module in a final reaction volume of 8 ul and NucleoMag NGS Clean-up and Size select magnetic beads were used for all DNA purification and size selection steps. For the 9-cell-mixture plates, clean up of the PCR product was performed with 2X0.7-0.9 bead/DNA ratio. For the single cell and mRNA mixture plates, two different clean up ratios for the PCR product were used (0.8 followed by 0.9). The choice of clean-up ratio was optimized from the QC results of the 9-cell-mixture data and the SORT-seq protocols.
identifiers.namespace: GEO
identifiers.id: GSM3305230