GSM3665696: WTCHG_380869_254273: M38+ Spleen single cell; Mus musculus; RNA-Seq
Extraction and reverse transcription was as described by Picelli et al. (Nature Protocols, 2014). Briefly, single cells were lysed with 2ul buffer containing 0.2% (vol/vol) Triton X-100 and 2Uµ-1 Rnase inhibitorand mixed 1µl oligo-dT primer and 1µl of dNTP mix. Samples were incubated at 72°C for 3 mins.  Reverse transcription mix containing SuperScript II (Invitrogen) reverse transcriptase 100 U, RNAase inhibitor 10 U, SuperScript II first-strand buffer, DTT 5mM, betaine 1 M, MgCl2 6mM, TSO 1 µM and nuclease free water, was added to the samples. Samples where incubated at 45°C for 90mins followed by 10 cycles of 50°C for 2mins and 42°C for 2 min. Library preparation of purified amplified cDNA was performed using Nextera XT library preparation (Illumina, AUS). Following QC, 150 pg of cDNA was tagmented (simultaneously fragmented with adaptors inserted) using Nextera transposons. Molecular barcodes were incorporated during 12 cycles of amplification followed by purification using AMPure XP. The libraries passed a quality checkpoint (Qubit and Bioanalyser HS DNA) prior to normalization and pooling before loading onto the HiSeq 2500 (Illumina, AUS) for paired end sequencing.
GEO
GSM3665696