GSM4120159: low response profile cell 16; Homo sapiens; RNA-Seq
Laser-captured single-cells were catapulted into 0.2ml domed caps tube (strips) containing 4.3 µl of lysis buffer (0,2% w/v Tween 20, 1.5U/µl Promega RNAseIN Plus). Tubes where kept on ice and frozen in dry ice directly after collection. Samples were thawed on ice and 2.43μl of a mix containing 10,000 molecules Life Technologies ERCC spike-In Mix 1, 2.5mM dNTPs, and 3.7µM single barcoded reverse transcription primer was added to each tube, incubated for 10 min at room temperature, 3 min at 70°C and kept at 10°C. Immediately after lysis, 8.27 µl 1.8X reverse transcription buffer (Invitrogen), 4.53mM DTT, 1.8M Betaïne, 10.8 mM MgCl2, 3.62 µM template switching oligonucleotide, 0.6U/µl Promega RNAse IN Plus, 7.5 U/µl Invitrogen SuperScript II reverse transcriptase were added and samples were reverse transcribed 10 min at 25°C, 90 min at 42°C, 15 min at 70°C and kept at 10°C until PCR amplification. Samples were PCR amplified (KAPA HiFi Hotstart) 20 cycles in 31 μl with 0.8µM forward PCR primer, 0.8µM biotinylated reverse PCR primer. Samples were incubated 3 min at 98°C followed by 20 cycles at 98°C for 20 sec, 64°C for 15 sec, 72°C for 6 min followed by a final extension for 5 min at 72°C. After PCR amplification, samples were pooled and purified with 0.8X Beckman Coulter SPRIselect beads, washed once with 85% EtOH and eluted in 30 μl. Batches of barcoded cDNAs were pooled, purified with SPRIselect beads (0.8x) and eluted in 12µl. Nine µl (typically 10 – 20 ng of cDNA) were used for tagmentation and Illumina library preparation as described in Arguel MJ et al. NAR 2017
GEO Accession
GSM4120159