GSM2653636: T96s1_G12; Homo sapiens; RNA-Seq
Poly(A)+ mRNA from isolated single cells were converted to cDNA decorated with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs) using a template-switching reverse transcriptase. Decorated cDNA from multiple cells were then pooled, amplified and prepared for multiplexed sequencing using a modified transposon-based fragmentation approach that enriches for 3’ ends and preserves strand information. Frozen cells were thawed for 5 minutes at room temperature and cell lysis was enhanced by a treatment with proteinase K (200μg/mL;Ambion) followed by RNA desiccation to inactivate the proteinase K and simultaneously reduce the reaction volume (50 °C for 15 in sealed plate, then 95 °C for 10 min with seal removed ). Diluted ERCC RNA Spike-In Mix (1μl of 1:107 ; Life Technologies) was added to each well. Template switching reverse transcription reaction was carried out using Maxima H Minus Reverse Transcriptase (Thermo Scientific). Following the template switching reaction, cDNA from 384 wells were pooled together, and then purified and concentrated using a single DNA Clean & Concentrator-5 column (Zymo Research). Pooled cDNAs were treated with Exonuclease I (New England Biolabs) and then amplified by single primer PCR using the Advantage 2 Polymerase Mix (Clontech). Full length cDNAs were purified with Agencourt AMPure XP magnetic beads (0.6x, Beckman Coulter) and quantified on the Qubit 2.0 Flurometer using the dsDNA HS Assay (Life Technologies). Full-length cDNA was then used as input to the Nextera XT library preparation kit (Illumina) according to the manufacturer’s protocol, with the exception that the i5 primer was replaced by our P5NEXTPT5 primer (Integrated DNA Technologies). The resulting sequencing library was purified with Agencourt AMPure XP magnetic beads (0.6x, Beckman Coulter), size selected (300-800bp) on a E-Gel EX Gel, 2% (Life Technologies), purified using the QIAquick Gel Extraction Kit (Qiagen) and quantified on the Qubit 2.0 Flurometer using the dsDNA HS Assay (Life Technologies). Libraries were sequenced on Illumina Hiseq paired-end flow cells with 17 cycles on the first read to decode the well barcode and UMI, a 9 cycle index read to decode the i7 Nextera barcode that serves as a plate / pool barcode, and finally a 46 cycle second read to sequence the cDNA.
GEO Accession
GSM2653636