GSM5151260: RBG26735_Old_SEC_058; Mus musculus; RNA-Seq
Single endosteal and central marrow stromal cells were directly sorted into individual wells of a 96-well PCR plate containing 2.3 µl of 0.2% Triton-X100 (Sigma-Aldrich, 93443) and 1U of Superase-In RNase Inhibitor (Ambion). Of note, information regarding expression of surface markers was recorded for each cell. Cells were frozen immediately at -80C until further processing. After thawing on ice, 2 µl of annealing mixture (1 µM oligodT –IDT-, 5mM each dNTPs and a dilution 1:6,000,000 of ERCC RNA Spike-In Mix -Invitrogen-) was added followed by incubation at 72C for 3 minutes. Then 5.7 µl of Reverse Transcription mix (3.5 U/µl of Maxima H minus retrotranscriptase –ThermoFisher-, 0.88 U/µl of Superase-In RNase Inhibitor, 1.75X Maxima RT Buffer, 3.5 µM TSO – Qiagen- and 13.15% PEG 8000 –Sigma-Aldrich-) was added and the mixture was incubated at 42 C for 90 min, followed by 70 C for 15 min. cDNA was further amplified by adding 40 µl of PCR mix (0.03 U/µl of Terra PCR direct polymerase –Takara Bio-, 1.25X Terra PCR Direct Buffer, 0.25 µM IS PCR primer –IDT-). PCR was as follows: 3 min at 98 °C for initial denaturation followed by 21 cycles of 15 s at 98 °C, 30 s at 65 °C, 4 min at 68 °C. Final elongation was performed for 10 min at 72 °C.  Sequences of oligodT, TSO and IS PCR primers were as previously described (PMID: 24385147). Following preamplification, all samples were purified using Ampure XP beads (Beckman Coulter) at a ratio of 1:0.6 with a final elution in 25 µl of EB Buffer (Qiagen). The cDNA was then quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher). Size distributions were checked on high-sensitivity DNA chips (Agilent Bioanalyzer). Samples were used to construct Nextera XT libraries (Illumina) from 100 pg of preamplified cDNA. Libraries were purified and size selected (0.5X-0.7X) using Ampure XP beads. Then, libraries were quantified using KAPA qPCR quantification kit (KAPA Biosystems), pooled and sequenced in an Illumina HiSeq 4000 instrument.