TITLE: Single Cell Sequencing 10x Chromium 5’ VDJ
AUTHORS: Travis Dawson, Robert Marvin, Grace Chung

[DESCRIPTION]
Single cell RNA sequencing technique (scRNA-Seq) obtains gene expression profiles of individual cells for analysis, as opposed to comparing averaged gene expression signals between bulk samples of cells. The ability to examine transcriptional changes between individual cells at a high resolution uniquely define rare cell populations, identify heterogeneity within cell populations, investigate cell population dynamics in depth over time, and even interrogate cell signaling pathways.

ScRNA sequencing enables the exploration of the cellular heterogeneity from the various biospecimen tissues; tumor, biopsy samples and organoid cells. To profile the immune repertoire of cells, full-length (5’ UTR to constant region), paired T-cell receptor (TCR) and/or B-cell receptor (BCR) transcripts from 500-10,000 individual cells per sample can be assessed. A pool of approximately 750,000 barcodes are sampled separately to index each cell’s transcriptome and antigen specificity. It is done by partitioning thousands of cells into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Libraries are generated and sequenced and 10x Barcodes are used to associate individual reads back to the individual partitions.

[STEPS]
SECTION: GEM Generation and RT Reaction
1. Prepare master mix on ice. Pipette mix 15x and centrifuge briefly.

 

SECTION: GEM Generation and RT Reaction
2. Add 37.2 μl Master Mix into each tube of a PCR 8-tube strip on ice.
SECTION: GEM Generation and RT Reaction
3. Assemble Chromium Next GEM Chip G:

 

SECTION: GEM Generation and RT Reaction
4. Load 50% Glycerol into Unused Chip Wells (if <8 samples per chip): i. 70 ul to unused wells in row labeled 1. ii. 50 ul to unused wells in row labeled 2. iii. 45 ul to unused wells in row labeled 3.
SECTION: GEM Generation and RT Reaction
5. Prepare Master Mix + Cell Suspension. Refer to the Cell Suspension Volume Calculator Table. Add the appropriate volume of nuclease-free water and corresponding volume of single cell suspension to Master Mix for a total of 75 ul in each tube. Gently pipette mix the cells suspension before adding to the Master Mix.
SECTION: GEM Generation and RT Reaction
6. Gently pipette mix the Master Mix + Cell Suspension and using the same pipette tip, dispense 70 ul Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.
SECTION: GEM Generation and RT Reaction
7. Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec. Centrifuge the Gel Bead strip for ~5 seconds. Confirm there are no bubbles at the bottom of the tubes and the liquid levels are even. Place the Gel Bead strip back in the holder. Secure the holder lid.
SECTION: GEM Generation and RT Reaction
8. Puncture the foil seal of the Gel Bead tubes. Slowly aspirate 50 ul Gel Beads. Dispense into the wells in row labeled 2 without introducing bubbles. Wait 30 seconds.
SECTION: GEM Generation and RT Reaction
9. Dispense 45 ul Partitioning Oil into the wells in row labeled 3 from a reagent reservoir. Failure to add Partitioning Oil to the top row labeled 3 will prevent GEM generation and can damage the Chromium Controller.
SECTION: GEM Generation and RT Reaction
10. Attach 10x gasket (notch on top left)
SECTION: GEM Generation and RT Reaction
11. Place the assembled chip with the gasket into the tray of the Chromium Controller, ensuring the chip stays horizontal. Close the tray and press the play button to begin run (18 minutes). Complete next steps during run.
SECTION: GEM Generation and RT Reaction
12. Place a PCR 8-tube strip on ice.
SECTION: GEM Generation and RT Reaction
13. When chip run is complete, press the eject button of the Controller to remove the chip. Discard the gasket, open chip holder, and fold the lid back until it clicks to expose the wells at a 45-degree angle.
SECTION: GEM Generation and RT Reaction
14. Transfer 100 μl of GEMs into PCR strip tube. Pipette slowly. It should take ~20 seconds to pipette GEMs.
SECTION: GEM Generation and RT Reaction
15. If multiple chips are run back-to-back, cover the GEM-containing strip tube and place on ice for no more than 1 hour.
SECTION: GEM Generation and RT Reaction
16. Run the following thermocycler program: 

 
NOTE: Store at 4°C for up to 72 h or at −20°C for up to a week, or proceed to the next step.
SECTION: Post GEM Incubation Cleanup
17. Add 125 ul Recovery Agent to each sample at room temperature. DO NOT pipette mix or vortex the biphasic mixture. Gently invert tube 10x to mix. Centrifuge briefly.
SECTION: Post GEM Incubation Cleanup
18. Slowly remove and discard 125 ul Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
SECTION: Post GEM Incubation Cleanup
19. Prepare Dynabeads Cleanup Mix:

 

SECTION: Post GEM Incubation Cleanup
20. Vortex and add 200 μl of Dynabeads Cleanup Mix to each sample. Pipette mix 5x (pipette set to 200 ul).
SECTION: Post GEM Incubation Cleanup
21. Incubate 10 min at room temperature.
SECTION: Post GEM Incubation Cleanup
22. Prepare Elution Solution I. Vortex and centrifuge briefly:

 

SECTION: Post GEM Incubation Cleanup
23. At the end of 10 min incubation, place on the 10x Magnetic Separator, high position (Magnet – High) until the solution clears.
SECTION: Post GEM Incubation Cleanup
24. Remove the supernatant.
SECTION: Post GEM Incubation Cleanup
25. Add 300 ul freshly prepared 80% ethanol to the pellet while on the magnet – High. Wait 30 seconds.
SECTION: Post GEM Incubation Cleanup
26. Remove the ethanol.
SECTION: Post GEM Incubation Cleanup
27. Add 200 ul 80% ethanol to the pellet. Wait 30 seconds.
SECTION: Post GEM Incubation Cleanup
28. Remove the ethanol.
SECTION: Post GEM Incubation Cleanup
29. Centrifuge briefly. Place on the magnet – Low.
SECTION: Post GEM Incubation Cleanup
30. Remove the remaining ethanol.
SECTION: Post GEM Incubation Cleanup
31. Remove from the magnet. Immediately add 35.5 ul Elution Solution I to avoid clumping.
SECTION: Post GEM Incubation Cleanup
32. Pipette mix 15x (pipette set to 30 ul) without introducing bubbles.
SECTION: Post GEM Incubation Cleanup
33. Incubate 2 minutes at room temperature
SECTION: Post GEM Incubation Cleanup
34. Centrifuge briefly. Place on the magnet – Low until the solution clears.
SECTION: Post GEM Incubation Cleanup
35. Transfer 35 ul sample to a new tube strip.
SECTION: cDNA Amplification
36. Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.

 

SECTION: cDNA Amplification
37. Add 65 ul cDNA Amplification Reaction Mix to 35 ul sample. Pipette mix 15x (pipette set to 90 ul). Centrifuge briefly.
SECTION: cDNA Amplification
38. Incubate in a thermal cycler with the following protocol. NOTE: Samples can be stored at 4°C up to 72 hours or at -20°C for up to 1 week or proceed to the next step.

 

SECTION: cDNA Amplification
39. Ensure sample volume is 100 μl (bring up to 100 μl with Buffer EB if needed). Vortex to resuspend the SPRIselect reagent. Add 60 ul SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 ul).
SECTION: cDNA Amplification
40. Incubate 5 min at room temperature.
SECTION: cDNA Amplification
41. Place on the magnet – High until the solution clears (~5 minutes).
SECTION: cDNA Amplification
42. Remove the supernatant.
SECTION: cDNA Amplification
43. Add 200 ul of 80% ethanol to the pellet. Wait 30 sec.
SECTION: cDNA Amplification
44. Remove the ethanol.
SECTION: cDNA Amplification
45. Repeat steps 43 and 44 for a total of 2 washes.
SECTION: cDNA Amplification
46. Centrifuge briefly and place on the magnet – High.
SECTION: cDNA Amplification
47. Remove any remaining ethanol. Air dry for 2 minutes. DO NOT exceed 2 minutes as this will decrease elution efficiency.
SECTION: cDNA Amplification
48. Remove from magnet. Add 45.5 ul Buffer EB. Pipette mix 15x.
SECTION: cDNA Amplification
49. Incubate 2 min at room temperature.
SECTION: cDNA Amplification
50. Place the tube strip on the magnet – High until the solution clears (~2 minutes).
SECTION: cDNA Amplification
51. Transfer 45 ul sample to a new tube strip and *Label the top of tubes with "cDNA". NOTE: Store at 4°C for up to 72 hours or at −20°C for up to 4 weeks or proceed to the next step.
SECTION: cDNA QC &amp; Quantification
52. Run 1 ul of sample on Qubit dsDNA HS Assay.
SECTION: cDNA QC &amp; Quantification
53. Either run 1 ul of 1 ng/ul diluted sample on an Agilent Bioanalyzer High Sensitivity chip to determine fragment size.
SECTION: cDNA QC &amp; Quantification
54. OR run 2 ul of 1 ng/ul diluted sample on the Agilent Tapestation High Sensitivity D1000 ScreenTape to determine fragment size.

 

SECTION: Gene Expression Library Construction
55. Initiate the following thermocycler protocol to pre-cool the block.
 

SECTION: Gene Expression Library Construction
56. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly.

 



SECTION: Gene Expression Library Construction
57. Determine the volume for 50 ng mass of sample. Dispense the sample volume in a tube strip on ice. If the volume required for 50 ng is less than 20 μl, adjust the total volume of each sample to 20 μl with nuclease-free water. If the volume for 50 ng exceeds 20 μl, carry ONLY 20 μl sample into library construction.
SECTION: Gene Expression Library Construction
58. Aliquot 30 μl of Fragmentation Mix to each well, mix 15x and spin down before returning to ice, let cool for 1 minute.
SECTION: Gene Expression Library Construction
59. Transfer to pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.
SECTION: Gene Expression Library Construction
60. After the cycle is complete, vortex to resuspend SPRIselect reagent. Add 30 ul SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 75 ul).
SECTION: Gene Expression Library Construction
61. Incubate 5 min at room temperature.
SECTION: Gene Expression Library Construction
62. Place on the magnet – High until the solution clears (~5 minutes). DO NOT discard the supernatant.
SECTION: Gene Expression Library Construction
63. Transfer 75 ul supernatant to a new tube strip.
SECTION: Gene Expression Library Construction
64. Vortex to resuspend SPRIselect reagent. Add 10 ul SPRIselect reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 80 ul).
SECTION: Gene Expression Library Construction
65. Incubate 5 min at room temperature.
SECTION: Gene Expression Library Construction
66. Place on the magnet – High until the solution clears.
SECTION: Gene Expression Library Construction
67. Remove 80 ul supernatant, making sure not to discard any beads.
SECTION: Gene Expression Library Construction
68. Add 125 ul 80% ethanol to the pellet. Wait 30 seconds.
SECTION: Gene Expression Library Construction
69. Remove the ethanol.
SECTION: Gene Expression Library Construction
70. Repeat steps 68 and 69 for a total of 2 washes.
SECTION: Gene Expression Library Construction
71. Centrifuge briefly and place on the magnet – Low until the solution clears. Remove remaining ethanol. DO NOT over dry to ensure maximum elution efficiency.
SECTION: Gene Expression Library Construction
72. Remove from the magnet. Add 50.5 ul Buffer EB to each sample. Pipette mix 15x.
SECTION: Gene Expression Library Construction
73. Incubate 2 min at room temperature.
SECTION: Gene Expression Library Construction
74. Place on the magnet – High until the solution clears.
SECTION: Gene Expression Library Construction
75. Transfer 50 ul sample to a new tube strip.
SECTION: Gene Expression Library Construction
76. Prepare Adaptor Ligation Mix on ice. Pipette mix and centrifuge briefly.

 

SECTION: Gene Expression Library Construction
77. Add 50 μl of Adaptor Ligation Mix to 50 ul sample. Pipette mix 15x (pipette set to 90 ul). Centrifuge briefly.
SECTION: Gene Expression Library Construction
78. Incubate in a thermal cycler with the following protocol.

 

SECTION: Gene Expression Library Construction
79. Vortex to resuspend SPRIselect Reagent. Add 80 ul SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 ul).
SECTION: Gene Expression Library Construction
80. Incubate 5 min at room temperature.
SECTION: Gene Expression Library Construction
81. Place on the magnet – High until the solution clears.
SECTION: Gene Expression Library Construction
82. Remove the supernatant.
SECTION: Gene Expression Library Construction
83. Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
SECTION: Gene Expression Library Construction
84. Remove the ethanol.
SECTION: Gene Expression Library Construction
85. Repeat steps 83 and 84 for a total of 2 washes.
SECTION: Gene Expression Library Construction
86. Centrifuge briefly. Place on the magnet – Low.
SECTION: Gene Expression Library Construction
87. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
SECTION: Gene Expression Library Construction
88. Remove from the magnet. Add 30.5 ul Buffer EB. Pipette mix 15x.
SECTION: Gene Expression Library Construction
89. Incubate 2 min at room temperature.
SECTION: Gene Expression Library Construction
90. Place on the magnet – Low until the solution clears.
SECTION: Gene Expression Library Construction
91. Transfer 30 ul sample to a new tube strip.
SECTION: Gene Expression Library Construction
92. Choose the appropriate sample index set to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x sample index name (PN-2000240 Single Index Plate T Set A well ID) used.
SECTION: Gene Expression Library Construction
93. Prepare Sample Index PCR Mix:

 

SECTION: Gene Expression Library Construction
94. Add 60 ul Sample Index PCR Mix to 30 ul sample.
SECTION: Gene Expression Library Construction
95. Add 10 ul of an individual Single Index to each well and record the well ID used. Pipette mix 5x (pipette set to 90 ul). Centrifuge briefly.
SECTION: Gene Expression Library Construction
96. Incubate in a thermal cycler with the following protocol. NOTE: Store at 4°C for up to 72 hours or at −20°C for up to 4 weeks or proceed to the next step.

 

SECTION: Gene Expression Library Construction
97. Vortex to resuspend the SPRIselect reagent. Add 60 ul SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 ul).
SECTION: Gene Expression Library Construction
98. Incubate 5 minutes at room temperature.
SECTION: Gene Expression Library Construction
99. Place on the magnet – High until the solution clears (~2 minutes).
SECTION: Gene Expression Library Construction
100. Discard the supernatant
SECTION: Gene Expression Library Construction
101. Add 200 ul 80% ethanol to the pellet. Wait 30 seconds.
SECTION: Gene Expression Library Construction
102. Remove the ethanol.
SECTION: Gene Expression Library Construction
103. Repeat steps 101 and 102 for a total of 2 washes.
SECTION: Gene Expression Library Construction
104. Centrifuge briefly. Place on the magnet – Low.
SECTION: Gene Expression Library Construction
105. Remove remaining ethanol.
SECTION: Gene Expression Library Construction
106. Remove from the magnet. Immediately add 35.5 ul Buffer EB. Pipette mix 15x
SECTION: Gene Expression Library Construction
107. Incubate 2 minutes at room temperature.
SECTION: Gene Expression Library Construction
108. Centrifuge briefly. Place on the magnet – Low.
SECTION: Gene Expression Library Construction
109. Transfer 35 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hours or -20°C for long-term storage.
SECTION: V(D)J Target Enrichment from cDNA
110. Add 33ul of Nuclease-Free water to PCR tube on ice.
SECTION: V(D)J Target Enrichment from cDNA
111. Add 2ul of cDNA Amp product to water.
SECTION: V(D)J Target Enrichment from cDNA
112. Prepare Target Enrichment 1 master mix on ice.

 

SECTION: V(D)J Target Enrichment from cDNA
113. Add 65ul of master mix to each sample, mix 5x on ice.
SECTION: V(D)J Target Enrichment from cDNA
114. Run the following thermocycler conditions. NOTE: Samples can be stored at 4°C up to 72 hours before the next step.
 

SECTION: V(D)J Target Enrichment from cDNA
115. Vortex to resuspend SPRIselect Reagent. Add 80 ul SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 ul).
SECTION: V(D)J Target Enrichment from cDNA
116. Incubate 5 min at room temperature.
SECTION: V(D)J Target Enrichment from cDNA
117. Place on the magnet – High until the solution clears.
SECTION: V(D)J Target Enrichment from cDNA
118. Remove the supernatant.
SECTION: V(D)J Target Enrichment from cDNA
119. Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
SECTION: V(D)J Target Enrichment from cDNA
120. Remove the ethanol.
SECTION: V(D)J Target Enrichment from cDNA
121. Repeat steps 10 and 11 for a total of 2 washes.
SECTION: V(D)J Target Enrichment from cDNA
122. Centrifuge briefly. Place on the magnet – Low.
SECTION: V(D)J Target Enrichment from cDNA
123. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
SECTION: V(D)J Target Enrichment from cDNA
124. Remove from the magnet. Add 30.5 ul Buffer EB. Pipette mix 15x.
SECTION: V(D)J Target Enrichment from cDNA
125. Incubate 2 min at room temperature.
SECTION: V(D)J Target Enrichment from cDNA
126. Place on the magnet – Low until the solution clears.
SECTION: V(D)J Target Enrichment from cDNA
127. Transfer 30 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hours or -20°C up to 1 week before the next step.
SECTION: V(D)J Target Enrichment from cDNA
128. Prepare Target Enrichment 2 master mix on ice.

 

SECTION: V(D)J Target Enrichment from cDNA
129. Add 65ul of master mix to each sample, mix 5x on ice.
SECTION: V(D)J Target Enrichment from cDNA
130. Run the following thermocycler conditions. NOTE: Samples can be stored at 4°C up to 72 hours before the next step.
 

SECTION: V(D)J Target Enrichment from cDNA
131. Vortex to resuspend SPRIselect Reagent. Add 50 ul SPRIselect Reagent (0.8X) to each sample and pipette mix 15x (pipette set to 145 ul).
SECTION: V(D)J Target Enrichment from cDNA
132. Incubate 5 min at room temperature.
SECTION: V(D)J Target Enrichment from cDNA
133. Place on the magnet – High until the solution clears. DO NOT discard the supernatant.
SECTION: V(D)J Target Enrichment from cDNA
134. Transfer 145 μl supernatant to a new tube strip.
SECTION: V(D)J Target Enrichment from cDNA
135. Add 30 μl of SPRIselect Reagent, mix 15x, incubate 5 min.
SECTION: V(D)J Target Enrichment from cDNA
136. Place on the magnet – High for 5 min., then remove 170 μl of supernatant.
SECTION: V(D)J Target Enrichment from cDNA
137. Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
SECTION: V(D)J Target Enrichment from cDNA
138. Remove the ethanol.
SECTION: V(D)J Target Enrichment from cDNA
139. Repeat steps 28 and 29 for a total of 2 washes.
SECTION: V(D)J Target Enrichment from cDNA
140. Centrifuge briefly. Place on the magnet – Low.
SECTION: V(D)J Target Enrichment from cDNA
141. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
SECTION: V(D)J Target Enrichment from cDNA
142. Remove from the magnet. Add 45.5 ul Buffer EB. Pipette mix 15x.
SECTION: V(D)J Target Enrichment from cDNA
143. Incubate 2 min at room temperature.
SECTION: V(D)J Target Enrichment from cDNA
144. Place on the magnet – Low until the solution clears.
SECTION: V(D)J Target Enrichment from cDNA
145. Transfer 45 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hrs. or -20°C up to 1 week before the next step.
SECTION: V(D)J Target Enrichment Library Construction
146. Initiate following thermocycler protocol to pre-cool block:
 

SECTION: V(D)J Target Enrichment Library Construction
147. Prepare Fragmentation Mix on ice
 

SECTION: V(D)J Target Enrichment Library Construction
148. Determine the volume for 50 ng mass of sample (see example calculation at step 3.3). Dispense the sample volume in a tube strip on ice. If the volume required for 50 ng is less than 20 μl, adjust the total volume of each sample to 20 μl with nuclease-free water. If the volume for 50 ng exceeds 20 μl, carry ONLY 20 μl sample into library construction.
SECTION: V(D)J Target Enrichment Library Construction
149. Aliquot 30 μl of Fragmentation Mix to each well, mix 15x and spin down before returning to ice, let cool for 1 minute.
SECTION: V(D)J Target Enrichment Library Construction
150. Transfer to pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.
SECTION: V(D)J Target Enrichment Library Construction
151. Prepare Adaptor Ligation Mix on ice.
 

SECTION: V(D)J Target Enrichment Library Construction
152. Remove tube strip containing Fragmentation Rxn from the thermocycler.
SECTION: V(D)J Target Enrichment Library Construction
153. Add 50 μl of Adaptor Ligation Mix to each sample, mix 15x, and spin briefly.
SECTION: V(D)J Target Enrichment Library Construction
154. Run the following thermocycler program:
 

SECTION: V(D)J Target Enrichment Library Construction
155. Remove tube from thermocycler. Add 80 μl of SPRIselect Reagent, mix 15x, incubate 5 minutes, complete next step during incubation.
SECTION: V(D)J Target Enrichment Library Construction
156. Prepare Sample Index PCR Mix on ice.
 

SECTION: V(D)J Target Enrichment Library Construction
157. Place on the magnet – High until the solution clears.
SECTION: V(D)J Target Enrichment Library Construction
158. Remove the supernatant...
SECTION: V(D)J Target Enrichment Library Construction
159. Add 200 ul 80% ethanol to the pellet. Wait 30 sec.
SECTION: V(D)J Target Enrichment Library Construction
160. Remove the ethanol.
SECTION: V(D)J Target Enrichment Library Construction
161. Repeat steps 159 and 160 for a total of 2 washes.
SECTION: V(D)J Target Enrichment Library Construction
162. Centrifuge briefly. Place on the magnet – Low.
SECTION: V(D)J Target Enrichment Library Construction
163. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
SECTION: V(D)J Target Enrichment Library Construction
164. Remove from the magnet. Add 30.5 ul Buffer EB. Pipette mix 15x.
SECTION: V(D)J Target Enrichment Library Construction
165. Incubate 2 min at room temperature.
SECTION: V(D)J Target Enrichment Library Construction
166. Place on the magnet – Low until the solution clears.
SECTION: V(D)J Target Enrichment Library Construction
167. Transfer 30 ul sample to a new tube strip.
SECTION: V(D)J Target Enrichment Library Construction
168. Add 60 μl of Sample Index PCR Mix to each sample
SECTION: V(D)J Target Enrichment Library Construction
169. Add 10 μl of an individual sample index (Single Index Plate T Set A) to each well and record their assignment.
SECTION: V(D)J Target Enrichment Library Construction
170. Mix by pipetting 15x and spin briefly. Run the following thermocycler program:NOTE: Samples can be stored at 4°C up to 72 hours before the next step
 

SECTION: V(D)J Target Enrichment Library Construction
171. Vortex to resuspend SPRIselect reagent. Add 80 ul SPRIselect reagent to each sample. Pipette to mix 15x.
SECTION: V(D)J Target Enrichment Library Construction
172. Incubate 5 minutes at room temperature.
SECTION: V(D)J Target Enrichment Library Construction
173. Place on the magnet – High until the solution clears (~2 minutes).
SECTION: V(D)J Target Enrichment Library Construction
174. Discard the supernatant
SECTION: V(D)J Target Enrichment Library Construction
175. Add 200 ul 80% ethanol to the pellet. Wait 30 seconds.
SECTION: V(D)J Target Enrichment Library Construction
176. Remove the ethanol.
SECTION: V(D)J Target Enrichment Library Construction
177.  Repeat steps 175 and 176 for a total of 2 washes.
SECTION: V(D)J Target Enrichment Library Construction
178. Centrifuge briefly. Place on the magnet – Low.
SECTION: V(D)J Target Enrichment Library Construction
179. Remove remaining ethanol.
SECTION: V(D)J Target Enrichment Library Construction
180. Remove from the magnet. Immediately add 35.5 ul Buffer EB. Pipette mix 15x
SECTION: V(D)J Target Enrichment Library Construction
181. Incubate 2 minutes at room temperature.
SECTION: V(D)J Target Enrichment Library Construction
182. Centrifuge briefly. Place on the magnet – Low.
SECTION: V(D)J Target Enrichment Library Construction
183. Transfer 35 ul sample to a new tube strip. NOTE: Samples can be stored at 4°C up to 72 hours or -20°C for long-term storage.
SECTION: LIBRARY QUANTIFICATION
184. Qubit: Run 1 ul sample at 1:5 dilution on the Qubit dsDNA HS Assay Kit
SECTION: LIBRARY QUANTIFICATION
185. Bioanalyzer/Tapestation:
EITHER Run 1 ul sample diluted to 3 ng/ul on the Agilent Bioanalyzer High Sensitivity DNA chip to determine fragment size. Lower molecular weight product (<150 bp) may be present. This does not affect sequencing.
OR Run 2 ul sample diluted to 1 ng/ul on the Agilent Tapestation High Sensitivity D1000 ScreenTape to determine fragment size.
 

SECTION: LIBRARY QUANTIFICATION
186.3. Make enough Quantification Master Mix for the DNA dilutions per sample and the DNA Standards (plus 10% excess) using the guidance for 1 reaction volume below:
 

SECTION: LIBRARY QUANTIFICATION
186.2. Dilute 1 ul sample with deionized water to appropriate dilutions that fall within the linear detection range of the KAPA Library Quantification Kit for Illumina Platforms. (For more accurate quantification, make the dilution(s) in duplicate).
SECTION: LIBRARY QUANTIFICATION
186.1. Thaw KAPA Library Quantification Kit for Illumina Platforms
SECTION: LIBRARY QUANTIFICATION
186. qPCR


SECTION: LIBRARY QUANTIFICATION
186.4. Dispense 16 ul Quantification Master Mix for sample dilutions and DNA Standards into a 96 well PCR plate.
SECTION: LIBRARY QUANTIFICATION
186.5. Incubate in a thermal cycler with the following protocol.
 

SECTION: LIBRARY QUANTIFICATION
186.6. Follow the manufacturer’s recommendations for qPCR-based quantification. For library quantification for sequencer clustering, determine the concentration using the average size in the region of 175 – 1,000 bp for gene expression libraries.
SECTION: SEQUENCING
187. Sequencing Libraries: Chromium Single Cell V(D)J Enriched libraries, 5' Gene Expression libraries, and Cell Surface Protein libraries comprise standard Illumina paired-end constructs which begin with P5 and end with P7. These libraries include 16 bp 10x Barcodes encoded at the start of TruSeq Read 1. Sample index sequences are incorporated as the i7 index read for V(D)J Enriched and 5' Gene Expression libraries; as i7 index read N for Cell Surface Protein library. TruSeq Read 1, TruSeq Read 2, and Nextera Read 2 (Read 2N) are all standard Illumina sequencing primer sites. TruSeq Read 1 and TruSeq Read 2 are used in paired-end sequencing of V(D)J Enriched and 5' Gene Expression libraries. TruSeq Read 1 and Nextera Read 2 (Read 2N) are used for paired-end sequencing of Cell Surface Protein library. Sequencing these libraries produce a standard Illumina BCL data output folder.
 

SECTION: SEQUENCING
188. Illumina Sequencer Compatibility
The compatibility of the listed sequencers has been verified by 10x Genomics. Some variation in assay performance is expected based on sequencer choice.
 MiSeq
 NextSeq 500/550 (High Output)
 HiSeq 2500 (Rapid Run)
 HiSeq 3000.4000
 NovaSeq
SECTION: SEQUENCING
189. Sample Indices
Each sample index in the Chromium i7 Sample Index Kit (PN-120262) and Chromium i7 Sample Index Kit Plate N, Set A (PN-1000084) is a mix of 4 different sequences to balance across all 4 nucleotides. If multiple samples are pooled in a sequence lane, the sample index name (i.e. the Chromium i7 Sample Index plate well ID) is needed in the sample sheet used for generating FASTQs with “cellranger mkfastq”. If multiple libraries are pooled in a sequence lane, a separate sample index is needed with each library.
SECTION: SEQUENCING
190. Sequencing Depth & Run Parameters
 

SECTION: SEQUENCING
191. Library Pooling
The 3ʹ Gene Expression libraries maybe pooled for sequencing, taking into account the differences in cell number and per-cell read depth requirements between each library. Samples utilizing the same sample index should not be pooled together, or run on the same flow cell lane, as this would not enable correct sample demultiplexing.