GSM5770375: Hypothalamus309935; Mus musculus; RNA-Seq
Hypothalamus region of the mouse brain was isolated by laser capture microdissection (LCM) and collected in 96-well plate containing barcoded magnetic beads. The tissue was lysed in the well, allowing mRNA to be captured by oligonucleotides on the surface of the magnetic beads. In detail, each well was loaded with 50 μL 2X lysis buffer (Tris-HCl(pH7.5) 120 μL, LiCl 80 μL, 10% SDS 120 μL, EDTA 16 μL, 500 mM DTT 12 μL, DNase and RNase-free water 852 μL). The tissue was lysed on ice for 12 minutes. The beads were washed with 6X SSC (Invitrogen, Cat#AM9770) and then the suspension was transfered to 1.5 mL centrifugal tube. The supernatant was removed. The beads were re-suspended with 500 μL 6X SSC, and the supernatant was removed after centrifugation. The beads were re-suspended with 300 μL 50 mM Tris(pH8.0, Beyotime Biotec., Cat#ST780) and then remove the supernatant. The beads were re-suspended with 20 μL RT Mix(DNase and RNase-free water 16.16 μL， 5X Superscript II first-strand buffer 8 μL, betaine (Sigma, Cat#B0300) 8 μL, 10 mM dNTPs 4 μL, DTT (Beyotime Biotec., Cat#ST041) 0.2 μL, MgCl2 (Invitrogen, Cat#AM9530G) 0.24 μL, 50 μM TSO primer 0.4 μL, PrimeScript II Reverse Transcriptase (Takara, Cat#2690A) 2 μL, and RNase Inhibitor 1 μL). The captured mRNA underwent reverse transcription (RT) at 42 ℃ for 90 min. Remove supernatant after centrifugation. The beads were washed with 200 μL TE-SDS (10% SDS and TE(pH8.0, Invitrogen, Cat#AM9849)), 200 μL TE-TW (10% Tween-20 (Sigma, Cat#P7949) and TE(PH8.0)), and 200 μL 10 mM Tris (pH8.0) in turn and remove the supernatant. The beads were re-suspended with 200 μL exonuclease mix (DNase and RNase-free water 170 μL, 10X Exol Buffer 20 μL, and Exol (Annoron, Cat#M0293L) 10 μL) and then incubated at 37 ℃ for 60 min. The beads were washed with 200 μL TE-SDS, 200 μL TE-TW and 200 μL 10 mM Tris (pH8.0) in turn and remove the supernatant. Beads were distributed to 4 tubes. To every tube, 12.5 μL of PCR mix was added, which included 2X HiFi HotStart Readymix (Kapa Biosystems) and 0.5 μL 10 μM TSO-PCR primer. The PCR program was as follows: 98 ℃ for 3 min; 6 cycles of 98 ℃ 20 s, 65 ℃ 45 s, and 72 ℃ 6 min; 72 ℃  10 min and 4 ℃ hold. After pooling all PCR products, AMPure XP beads (Beckman Coulter) were used to purify the cDNA library. cDNA library was prepared following the guidance of Illumina Nextera XT DNA Library Preparation Kits.