GSM5287239: Rat blood vessel-control; Rattus norvegicus; RNA-Seq
1.1 GEM Generation & Barcoding 1.1.1 Prepare Master Mix a. Prepare Master Mix on ice. Pipette mix 15x and centrifuge briefly. b. Add 33.4 μl Master Mix into each tube of a PCR 8-tube strip on ice. c. Assemble Chromium Chip B in a 10x Chip Holder. d. Dispense 50% Glycerol Solution into Unused Chip Wells i. 75 μl to unused wells in row labeled 1. ii. 40 μl to unused wells in row labeled 2. iii. 280 μl to unused wells in row labeled 3. e. Prepare Master Mix + Cell Suspension f. dispense 75 μl Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles. g. Prepare Gel Beads Snap the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec. h. Slowly aspirate 40 μl Gel Beads. Dispense into the wells in row labeled 2 without introducing bubbles. i. Dispense 280 μl Partitioning Oil into the wells in row labeled 3j. Attach 10x Gasket Run the Chromium Controller 1.1.2 Transfer GEMs a. Place a tube strip on ice. b. Slowly aspirate 100 μl GEMs from the lowest points of the Recovery Wells in the top row without creating a seal between the tips and the bottom of the wells. c. If multiple chips are run back-to-back, cap/ cover the GEM-containing tube strip and place on ice for no more than 1 h. d. GEM-RT Incubation e. Store at 4°C for up to 72 h or at −20°C for up to a week, or proceed to the next step. 2.2 Post GEM–RT Cleanup & cDNA Amplification 1.2.1 Post GEM-RT Cleanup – Dynabeads a. Add 125 μl Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Wait 60 sec. b. Slowly remove 125 μl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample. c. Prepare Dynabeads Cleanup Mix. d. Vortex and add 200 μl to each sample. Pipette mix 10x (pipette set to 200 μl). e. Incubate 10 min at room temperature. Pipette mix again at ~5 min after start of incubation to resuspend settled beads. f. Prepare Elution Solution I. Vortex and centrifuge briefly. g. At the end of 10 min incubation, place on a 10x Magnetic Separator-High position (magnet-High) until the solution clears. A white interface between the aqueous phase and Recovery Agent is normal. h. Remove the supernatant. i. Add 300 μl 80% ethanol to the pellet while on the magnet. Wait 30 sec. j. Remove the ethanol. k. Add 200 μl 80% ethanol to pellet. Wait 30 sec. l. Remove the ethanol. m. Centrifuge briefly. Place on the magnet-Low. n. Remove remaining ethanol. Air dry for 1 min. o. Remove from the magnet. Immediately add 35.5 μl Elution Solution I. p. Pipette mix (pipette set to 30 μl) without introducing bubbles. q. Incubate 2 min at room temperature. r. Place on the magnet-Low until the solution clears. s. Transfer 35 μl sample to a new tube strip. 1.2.2 cDNAAmplification a. Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly b. Add 65 μl cDNAAmplification Reaction Mix to 35 μl sample. c. Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly. d. Incubate in a thermal cycler with the following protocol. e. Store at 4°C for up to 72 h or proceed to the next step. 2.2.3 cDNA Cleanup – SPRIselect a. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 μl). b. Incubate 5 min at room temperature. c. Place on the magnet-High until the solution clears. d. Remove the supernatant. e. Add 200 μl 80% ethanol to the pellet. Wait 30 sec. f. Remove the ethanol. g. Repeat steps e and f for a total of 2 washes. h. Centrifuge briefly and place on the magnet-Low. i. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency. j. Remove from the magnet. Add 40.5 μl Buffer EB. Pipette mix 15x. k. Incubate 2 min at room temperature. l. Place the tube strip on the magnet-High until the solution clears. m. Transfer 40 μl sample to a new tube strip. n. Store at 4°C for up to 72 h or at −20°C for up to 4 weeks, or proceed to the next step. 2.3.1 Fragmentation, End Repair & A-tailing a. Prepare a thermal cycler with the following incubation protocol. . b. Vortex Fragmentation Buffer. Verify there is no precipitate. c. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly. d. Transfer ONLY 10 μl purified cDNA sample from cDNA Cleanup (step 2.3n) to a tube strip. e. Add 25 μl Buffer EB to each sample. f. Add 15 μl Fragmentation Mix to each sample. g. Pipette mix 15x (pipette set to 35 μl) on ice. Centrifuge briefly. h. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol. 2.3.2 Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect a. Vortex to resuspend SPRIselect reagent. Add 30 μl SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 75 μl). b. Incubate 5 min at room temperature. c. Place on the magnet-High until the solution clears. DO NOT discard supernatant. d. Transfer 75 μl supernatant to a new tube strip. e. Vortex to resuspend SPRIselect reagent. Add 10 μl SPRIselect reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 80 l). f. Incubate 5 min at room temperature. g. Place on the magnet-High until the solution clears. h. Remove 80 μl supernatant. DO NOT discard any beads. i. Add 125 μl 80% ethanol to the pellet. Wait 30 sec. j. Remove the ethanol. k. Repeat steps i and j for a total of 2 washes. l. Centrifuge briefly. Place on the magnet-Low until the solution clears. Remove remaining ethanol. DO NOT over dry to ensure maximum elution efficiency. m. Remove from the magnet. Add 50.5 μl Buffer EB to each sample. Pipette mix 15x. n. Incubate 2 min at room temperature. o. Place on the magnet-High until the solution clears. p. Transfer 50 μl sample to a new tube strip. 2.3.3 Adaptor Ligation a. Prepare Adaptor Ligation Mix. Pipette mix and centrifuge briefly. b. Add 50 μl Adaptor Ligation Mix to 50 μl sample. Pipette mix 15x (pipette set to 90 μl). Centrifuge briefly. c. Incubate in a thermal cycler with the following protocol. 2.3.4 Post Ligation Cleanup – SPRIselect a. Vortex to resuspend SPRIselect Reagent. Add 80 μl SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 μl). b. Incubate 5 min at room temperature. c. Place on the magnet-High until the solution clears. d. Remove the supernatant. e. Add 200 μl 80% ethanol to the pellet. Wait 30 sec. f. Remove the ethanol. g. Repeat steps e and f for a total of 2 washes. h. Centrifuge briefly. Place on the magnet-Low. i. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency. j. Remove from the magnet. Add 30.5 μl Buffer EB. Pipette mix 15x. k. Incubate 2 min at room temperature. l. Place on the magnet-Low until the solution clears. m. Transfer 30 μl sample to a new tube strip. 2.3.5 Sample Index PCR a. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x Sample Index name (PN-220103 Chromium i7 Sample Index Plate well ID) used. b. Prepare Sample Index PCR Mix. c. Add 60 μl Sample Index PCR Mix to 30 μl sample. d. Add 10 μl of an individual Chromium i7 Sample Index to each well and record the well ID used. Pipette mix 5x (pipette set to 90 μl). Centrifuge briefly. e. Incubate in a thermal cycler with the following protocol. 2.3.6 Post Sample Index PCR Double Sided Size Selection – SPRIselect a. Vortex to resuspend the SPRIselect reagent. Add 60 μl SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 μl). b. Incubate 5 min at room temperature. c. Place the magnet-High until the solution clears. DO NOT discard supernatant. d. Transfer 150 μl supernatant to a new tube strip. e. Vortex to resuspend the SPRIselect reagent. Add 20 μl SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 μl). f. Incubate 5 min at room