title: GSM2149298: naivecell_32; Homo sapiens; ncRNA-Seq
library_construction_protocol: Cells were lysed directly into the reaction tube. No RNA extraction was performed. Plates were pre-deposited with 3 µl of lysis buffer (0.13 % Triton-X-100, 4 u recombinant RNase Inhibitor, Takara). After single cell sorting, the plates were transferred to -80 °C for long storage. After thawing the plate, 1 µl of 5.8 S rRNA masking oligo (5 pmol) was added and the whole plate was incubated at 72 °C for 20 minutes.  Then, 2 µl of 3’ adaptor ligation reaction was added (20 pmol 3’ adaptor oligo, 8.33 % PEG 8000, 50 u T4 RNA Ligase 2, truncated KQ, NEB, 0.83 X T4 RNA ligase buffer, NEB, 4 u recombinant RNase Inhibitor, Takara) and reaction was incubated at 30 °C for 6 h followed by 4 °C for 10 hours.  Next, 3 µl of RT primer and free adaptor removal enzymes were added (200 pmol RT primer, 2.5 u Lambda exonuclease, NEB, 10 u 5’ deadenylase, NEB) and the reaction was incubated at 30 °C for 15 minutes followed by 37 °C for 15 minutes. Next, 2 µl of 5’ adaptor ligation reaction was added (45 pmol 5’ adaptor oligo, 0.68 mM Tris-buffered ATP, Thermo Fisher, 4 u T4 RNA ligase, Thermo Fisher,  0.23 X T4 RNA ligase buffer, NEB ) and the mix was incubated at 37 °C for 1 hour. Reverse transcription reaction was performed by adding 7 µl of RT reaction (1.28 X Taq DNA Polymerase PCR Buffer, Thermo Fisher, 8.33 mM DTT, 0.42 mM/each dNTP, 4 u recombinant RNase Inhibitor, 150 u Superscript II reverse transcriptase, Thermo Fisher). The PCR amplification is carried out by adding 35 µl of the reagents (0.94 X Phusion HF buffer, 1 u Phusion Hot Start II DNA Polymerase, Thermo Fisher, 0.12 mM/each dNTP,  1.89 µM RP1 primer) and incubating at 98 °C for 30 seconds followed by 13 cycles of 98 °C for 10 seconds, 60 °C for 30 seconds and 72 °C for 30 seconds and a final incubation at 72 °C for 5 minutes. At last, 1 µl of the amplified product is transferred to a fresh tube and the second PCR reaction is added (2 µM indexed primers, 0.2 µM RP1 primer, 1 X Phusion HF buffer, 0.5 u Phusion Hot Start II DNA Polymerase, Thermo Fisher,  0.2 mM/each dNTP) and incubating at 98 °C for 30 seconds followed by 13 cycles of 98 °C for 10 seconds, 67 °C for 30 seconds and 72 °C for 30 seconds and a final incubation at 72 °C for 5 minutes. The amplified libraries of single cells are then pooled and purified with DNA Clean & Concentrator™-5, Zymo research. All samples have been sequenced 1x 43 bp or 1x 51 bp using Illumina HiSeq 2500 instrument.
attributes.tag: GEO Accession
attributes.value: GSM2149298