title: GSM5343858: HYR__666332__20210412_mouse_cortex_a_S1; Mus musculus; RNA-Seq
library_construction_protocol: Cortex was separated from female P56 BL/6 mouse brains, immediately snap-frozen in liquid nitrogen and preserved in -80 °C. A ~1 cm3 frozen piece of mouse cortex tissue was transferred to 1 mL of ice-cold homogenisation buffer (320 mM Sucrose, 10 mM NaCl, 10 mM Tris-HCl pH 7.5, 3 mM MgAcetate, 1 mM DTT, 0.1 mM EDTA, 0.1% IGEPAL, 1X cOmplete Protease Inhibitor Cocktail) in a Dounce homogenizer mortar and thawed for 3 minutes. The tissue was homogenised with 10 strokes of pestle A and 5 strokes of pestle B until a homogeneous cell suspension was achieved. The resulting homogenate was filtered through a 40 μm Flowmi strainer. The mortar was washed with 1.65 mL of ice-cold homogenisation buffer to collect any remaining cells. 2.65 mL of ice-cold gradient medium was added to 1.65 mL of homogenate. 4 mL of 29% iodoxanol cushion (129.2 mM Sucrose, 77.5 mM KCl, 15.5 mM MgCl, 31 mM Tris-HCl pH 7.5, 29% Iodoxanol) was loaded into an ultracentrifuge. 5.3 mL of sample was gently layered on top of the 29% iodoxanol cushion without mixing both phases. Sample was centrifuged at 10 000 xg, 4°C for 30 minutes and the supernatant was gently removed without disturbing the nuclei pellet. Nuclei were resuspended in 100 μL of Nuclei buffer. Cells were resuspended in 85 μL of RT mix (1x Maxima RT Buffer, 0.9 mM dNTPs, 25 mM DTT, 1.3 mM GTP, 15 % Optiprep, 1.3 U/uL RNAse inhibitor, 15 U/uL Maxima hRT, 12.5 uM TSO, 4.4% PEG-8000). RT mix was co-encapsulated with 35 μL of freshly thawed HyDrop-RNA beads in RAN oil on the Onyx microfluidics platform. The resulting emulsion was collected in aliquots of 50 μL total volume and thermocycled according to the RT program (42 °C for 90 min., 11 cycles of [50 °C for 2 min., 42 °C for 2 min.], 85 °C for 5 min., followed by a final hold on 4 °C). 125 μL of recovery agent (20% PFO in HFE), 55 μL of GITC Buffer (5 M GITC, 25 mM EDTA, 50 mM Tris-HCl pH 7.4) and 5 μL of 1 M DTT was added to each separate aliquot of 50 μL thermocycled emulsion and incubated on ice for 5 minutes. 99 μL of Ampure XP beads was added to the aqueous phase and incubated for 10 minutes. Ampure beads were pelleted on a Nd magnet and washed twice with 80% EtOH. Elution was performed in 50 μL of EB-DTT-Tween (10 mM DTT, 0.1% Tween-20 in EB). Exonuclease treatment was performed by adding 3 μL of 10x NEBuffer 3.1, 3 μL of ExoI, and 4 μL of dH2O to 30 μL of eluted library. The ExoI reaction mix was incubated at 37 °C for 5 min., 80 °C for 1 min. followed by a final hold at 4 °C. 2 μL of 1 M DTT was added and a 0.8X Ampure XP purification was performed according to manufacturer's recommendations. cDNA was eluted in 40.5 μL of EB-DTT (10 mM DTT in EB) and added to ISPCR mix (40 μL library, 50 μL 2x KAPA HiFi, 10 μL 10 uM TSO-P primer). PCR cycling was performed according to the ISPCR program (95 °C for 3 min., 13 cycles of [98 °C for 20s, 63 °C for 20s, 72 °C for 3 min.], 72 °C for 5 min. followed by a final hold at 4 °C. 2 μL of 1M DTT was added and a 0.6x Ampure XP purification was performed according to manufacturer's recommendations. cDNA was eluted in 28.5 μL of EB-DTT. Final sequencing library was prepared according to the following customised NEB Ultra II FS protocol. 80 ng of amplified cDNA was fragmented in Ultra II fragmentation mix (26 μL of amplified cDNA, 7 μL of NEBNext Ultra II FS Reaction Buffer, 2 μL of NEBNext Ultra II FS Enzyme Mix) on the following thermocycling program: 37 °C for 10 min., 65 °C for 30 min. and a final hold at 4 °C. 15 μL of EB was added and a 0.8X Ampure purification was performed according to manufacturer's recommendation and eluted in 35 μL. Fragmented library was adapter-ligated in NEBNext Ultra II adapter ligation mix (35 μL of fragmented library, 30 μL of NEBNext Ultra II Ligation Master Mix, 1 μL of NEBNext Ligation Enhancer, 2.5 μL of NEBNext Adapter for Illumina) at 20 °C for 15 min., with 4 °C final hold. 28.5 μL of EB was added and a 0.8X Ampure purification was performed according to manufacturer's recommendation and eluted in 30 μL. Eluted library was amplified in PCR master mix (50 μL 2x KAPA HiFi, 10 μL 10 uM Hy-i7 primer, 10 μL 10 uM Hy-i5 primer, 30 μL eluted library) in the following thermocycling program: 95 °C for 3 min., 13 cycles of [98 °C for 20 s, 64 °C for 30 s, 72 °C for 30 s], 72 °C for 5 min. and a final hold at 4 °C. Sequencing-ready library was purified using a 0.8X Ampure purification and eluted in 30 μL of EB.
attributes.tag: GEO Accession
attributes.value: GSM5343858