title: GSM7211255: old 1; Mus musculus; RNA-Seq
library_construction_protocol: For 10X Genomics sequencing, ~ 30,000 HSCs were sorted into 1.5 ml tubes containing 500 µl of HBSS/2% FBS and transferred to the Columbia Genome Center Single Cell Analysis Core Facility for microfluidic cell processing, library preparation and sequencing. For SmartSeq2 sequencing, Single yHSC, AThi oHSC, ATmid oHSC, and ATlo oHSC were directly sorted into individual wells of a 96-well PCR plate in 2.3 µl of lysis buffer containing 0.2% Triton X-100 (Sigma-Aldrich) and 1U of Superase-In RNase Inhibitor (Ambion). Cells were frozen immediately at -80°C until further processing. For 10X, ~10,000 HSCs were loaded for each sample and 1 sample was loaded per condition.  For SmartSeq2, after thawing on ice, 2 µl of an annealing mixture containing1 µM oligodT (IDT), 5 mM each dNTPs and a 1:6,000,000 dilution of ERCC RNA Spike-In Mix (Invitrogen) was added followed by incubation at 72°C for 3 min. Then 5.7 µl of a Reverse Transcription mix containing 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 a PCR mix containing 0.03 U/µl of Terra PCR direct polymerase (Takara Bio), 1.25x Terra PCR Direct Buffer, and 0.25 µM IS PCR primer (ID). 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. 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). For 10X, samples were sequenced using an Illumina HiSEQ 4000 instrument. For SmartSeq2, 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. Libraries were quantified using KAPA qPCR quantification kit (KAPA Biosystems), pooled and sequenced using an Illumina HiSEQ 4000 instrument.