title: GSM4492701: P2277_N724_S513; Mus musculus; RNA-Seq
library_construction_protocol: For replicate 1, full-length single-cell RNA-seq libraries were prepared using the Smart-seq2 protocol (Picelli et al., 2013) with minor modifications. Thymocytes were sorted into 96-well plates containing the lysis buffer (0.2% Triton-100, 1U/µl RNase inhibitor)Reverse transcription was performed using SuperScript II (ThermoFisher Scientific) in the presence of 1 μM oligo-dT30VN (IDT), 1 μM template-switching oligonucleotides (QIAGEN), and 1 M betaine. cDNA was amplified using the KAPA Hifi Hotstart ReadyMix (Kapa Biosystems) and IS PCR primer (IDT), with 25 cycles of amplification. For replicate 2, full-length single-cell RNA-seq libraries were prepared using the SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing (Takara Bio). All reactions were downscaled to one quarter of the original protocol and performed following thermal cycling manufacturer's conditions. Thymocytes were sorted into 96-well plates containing 2.5 µl of the Reaction buffer (1x Lysis Buffer, RNase Inhibitor 1U/µl). Reverse transcription was performed using 2.5 µl of the RT MasterMix (SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing, Takara Bio). cDNA was amplified using 8 µl of the PCR MasterMix (SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing, Takara Bio) with 25 cycles of amplification. Replicate 1 followed purification of amplified cDNA with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with Nextera XT indexes (Illumina). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sequencing of Nextera libraries was carried out using a HiSeq2000 (Illumina) to an average sequencing depth of 0.5 million reads per cell. Sequencing was carried out as paired-end (PE75) reads with library indexes corresponding to cell barcodes. Replicate 2 followed purification of amplified cDNA with Agencourt Ampure XP beads (Beckmann Coulter), product size distribution and quantity were assessed on a Bioanalyzer using a High Sensitivity DNA Kit (Agilent Technologies). A total of 140 pg of the amplified cDNA was fragmented using Nextera XT (Illumina) and amplified with double indexed Nextera PCR primers (IDT). Products of each well of the 96-well plate were pooled and purified twice with Agencourt Ampure XP beads (Beckmann Coulter). Final libraries were quantified and checked for fragment size distribution using a Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled sequencing of Nextera libraries was carried out using a HiSeq4000 (Illumina) to an average sequencing depth of 0.5 million reads per cell. Sequencing was carried out as paired-end (PE100) reads with library indexes corresponding to cell barcodes.
identifiers.namespace: GEO
identifiers.id: GSM4492701